Introduction to Lyophilization

Join Gregory Sacha, Ph.D. of Baxter BioPharma Solutions for an introduction to lyophilization and the anatomy of a lyophilizer. Presented in front of a laboratory-scale lyophilizer, Dr. Sacha demonstrates placement of a thermocouple, manual loading, and discusses process parameters and equipment design that affect lyophilization cycles. Hooke College of Applied Sciences offers a freeze drying course.


So, let’s start by first understanding what the steps of lyophilization are, and why we would do them. The basis for Lyophilization is that we need something lyophilized if we to extend its shelf life, for example, it is not stable in solution. Lyophilization allows us to remove ice or water, from a product without destroying our volatile molecules. Not necessarily volatile, but those that might be susceptible to high heat. So, these products are placed in a lyophilizer, cooled and frozen, and then a vacuum is established to remove ice as sublimation.

So, those steps will include first filling the vials with solution and then taking those vials and placing them into a lyophilizer, and then cooling the vials down to around -40 degrees C. That step is the freezing step.

Cool the vials, allow them to completely freeze to -40, let’s say about two hours. And then now we can initiate a vacuum, that vacuum might be around, let’s say 100 millitorr, and then depending on the properties of those in solution, meaning those thermal characteristic properties, we may be able to increase the shelf temperature to somewhere around -20 or even higher, while continuing to pull that vacuum. That stage is primary drying; that is where we are removing the bulk ice.

After we remove all of the bulk ice, it is now safe to increase the temperature of the product. It is safe because all of that frozen water has been removed.

Now we need to increase the temperature of the product to drive off the unfrozen water. That section is known as secondary drying.

There is also a step that may be used during the first freezing step—that step is known as annealing. That is where if a product may crystallize, we can encourage crystallization by increasing the temperature of the product and then allowing it to rest, without pulling a vacuum. That step is known as annealing. That annealing time allows time for molecular movement which can encourage crystallization of a crystallizing component, or even encourage growth of ice crystals.

So, our goal is then to fill vials, and here is an example of a filled vial, our goal is to, after freeze drying, maintain the same height and volume of the solution that was filled. As an example of a freeze dry product, with an acceptable appearance, that is what we hope to do. What we hope not to do is produce something like this; this is collapse. This occurs if, for instance, we don’t understand the thermal behavior of our product and we exceed critical temperatures during primary drying. That is what we want to avoid.

Something else you may noticed is that, unlike the solution formulation, this formulation has a stopper that is partially seated. You will see that this stopper has a single vent. That single vent allows for the escape of water vapor during the process.

Before we move on, let’s first understand a little bit about the lyophilizer itself.

I am going to bring you a little closer to the lyophilizer, this is a laboratory scale lyophilizer. You will see it has a door with another chamber on the front, I will talk to you about that in a minute. Inside, this is the product chamber. There are three shelves to a product chamber—for ease of demonstration during this session, I have raised the top two shelves so we have plenty of room.

If we look a little further down, we see the condenser. The condenser is where ice is removed as water vapor during the sublimation it is trapped on these coils in the condenser. These coils are kept at a temperature around -65 or -70, somewhere around there. So, let me raise this camera a little bit.

How does that water vapor get to the chamber down to the condenser? This is an important thing to remember, not all lyophilizers are created the same. These lyophilizers have what is known as a spool piece in between the product chamber and the condenser. That spool piece is like a net. I will rotate you around to another lyophilizer that I have with the side panel removed. What we see here, we see a little bit, this neck piece right here between the chamber and the condenser. That is the spool piece. That is important to remember because some lyophilizers do not have a spool piece. They may just have this product chamber, and right next to that product chamber where the shelves are placed is the condenser, meaning really cold coils. Those coils can influence the temperature of your product. That is neither a good nor bad, but it is something to be aware of when developing your process and transferring it. Other lyophilizers still may not have a neck, but just this wall between the chamber and the condenser, with a plate that raises and falls depending on the stage of the process.

Something else that we need to discuss is how do we cool these vials? Where does this cold temperature come from?

These shelves are hollow. They have a cooling fluid, or a heat transfer fluid, that rotates and flows through them. Something else that is different between different lyophilizers is how that fluid flows. On some shelves it flows in a serpentine pattern, up and down. Other shelves will flow in a spiral pattern, this is a bit exaggerated, I’m terrible at drawing, but it’s a spiral.

Why do we care? We care because that definitely will determine how our heat is distributed on shelf. Neither one has an advantage or disadvantage, it is just that we need to be aware of that because there is something that’s known as the edge effect on a shelf. When we have a full shelf full of vials, the vials on the inside part of the shelf, the inner portion, will be much cooler than those that are on the very edge. What comes into effect is the wall temperature, door temperature, how wide these channels are, and how well they cover the entire shelf. That is something we need to be aware of.

When we fill vials, we fill them on a tray, here is a manual operation in our lab. We have vials filled onto a tray, all stoppers are partially seated.

You will notice a bunch of wires. These wires lead to vials that are equipped with thermocouples, so that we can monitor the temperature of our product during the process. Here is a vial with a thermocouple placed inside it. What we try to do, since these thermocouples are point sensors, we try to align that point as close as we can in the center of the vial in the center bottom. We do that because as ice is removed, it is removed from the top down. The bottom is going to be the coldest, and that can provide us a measure of when our primary drying cycle is complete. It is not the best way to measure, but it is a possible measurement. It is also a way to determine how close we are to that failure point temperature for a product. You will notice that I have a thermocouple placed in the front, the middle, and center. Different people place them with different methods. The coldest area is going to be in the center, edge areas will tell you how warm it might be— the warmest temperature you might experience during the process.

How do we place these in the lyophilizer? On a tray, the tray has a ring around it, so we place it in the lyophilizer and slide this top portion forward as we push. Now the bottom of the tray and the vials make direct contact with the shelf. We can then plug the thermocouples into the different ports. This allows us to monitor the product temperature throughout the process.

There are other types of thermocouples that we need to be aware of, or temperature monitoring systems. We won’t go into all of the details here, but there is RGD, there is a thermocouple that we place directly into the vial, then there are also these wireless temperature sensors. This one happens to be from Tempress and you will see it has a large, not really that large, but a glass bottom to it. That bottom contains a crystal that vibrates. And that vibration or oscillation will directly translate into the temperature of our product.

One reason why we like these wireless sensors is that they can be steam sterilized so that they can be used in our production process and then we also don’t have all of these wires. One challenge of placing thermocouples in a manufacturing area is that we can negatively affect sterility assurance. In a production area we may only be able to test or monitor the vials that are closest to the front of the door so we don’t reach over and negatively affect sterility assurance. These wireless thermocouples allow us to place vials and temperature sensors along the line, they can be randomly placed on an entire shelf.

After we plug these thermocouples in, we close the door. Close the door to the condenser chamber and then start our process, remembering the first portion of it is freezing, the primary drying, and secondary drying.

Things we monitor during the process; primary drying. Primary drying we want to determine when the end of it is. That end is determined by one, when we completely remove ice from our vials, and the temperature of our product becomes similar to the temperature of our shelf. Another method, and probably a more dependable method, I say more dependable because this method represents what is going on across the entire shelf or across the entire shelves, and that is comparative pressure measurement. Within this lyophilizer there is a compacitance manometer for measuring the set point pressure, for example if we it for 100 millitorr it will show when it is at 100 millitorr. Another measurement of pressure is resistance pressure measurement, known as a pirani guage. This electrical resistance is affected by the level of water vapor in the chamber. When water vapor is high, the pressure recorded by the pirani gauge is much higher than the pressure recorded by the compacitance manometer. This provides us a measure of when all of the water vapor is removed from our product chamber. At that point, the Pirani gauge measurement will become very similar to the compacitance manometer measurement. That tells us we can now proceed to secondary drying.

There are two little steps here that I would like to touch on. One is when we remove water vapor, what else goes into the chamber to balance that pressure? Continually, throughout this process, there is a nitrogen bleed a small amount of nitrogen into the chamber that replaces the water vapor that has been removed. That means that when these vials are finally sealed, they are sealed under an environment of nitrogen.

The next portion that I would like to touch on is, what is this? What is this box here? There is something that we need to know during our process. That is, what is the final residual moisture of our product? We start looking at that toward the end of primary drying and then in the secondary drying because we want to be able to take samples during those steps that will represent the high level residual moisture, the medium, and the low. We take those samples, place them on stability, and examine the effect of residual moisture. That tells us how low we need to go.

Then once we know the level of residual moisture we need, we now need to know at what shelf temperature —how long do we need to hold it at that shelf temperature to reach our desired level of residual moisture. We do all of that by taking samples from the chamber. We want to do that without completely breaking the vacuum. One method of doing that is this thief sampler. This thief sampler has a door on the front, we can seal it into place, and there is a door on the back that goes directly into the chamber. We can pull a vacuum on this external box until we can open the inside door. When we do that we now have access to the internal part of the chamber.

We can then reach this arm, it may be difficult to see, but there is a little grabbing device on the end of the arm. We can reach in there, pull a sample, pull it out, seal it, and then have a sample captured at that point of the process representing the certain residual moisture. Close that.

At the end of the process, it is your choice now to whether you want to seal under vacuum or not. Sealing under vacuum means that we make sure there is still a vacuum in there when we compress our shelves to seal the stoppers. That is how stoppers are sealed. Shelves are raised using a button until the vials make contact with the shelf above and they are pressed to seal the stoppers.

There is one other item I would like to touch on and that is what really is the driving force for removing that water vapor? It is a common misconception that it is that vacuum pulls it out the vials. That is really not how it works. What we are doing during primary drying is adjusting the pressure in the chamber and the shelf temperature to obtain our desired product temperature. By doing that, we are establishing a pressure difference between there and the chamber, and also a temperature difference. That pressure difference, the vapor pressure of ice on the chamber is very low. We have a very low chamber pressure. Here, we have a much higher temperature to help increase the rate of sublimation. When that occurs there is no pressure differential. The vapor pressure of ice is much higher here, so we are removing water vapor and now it is being trapped at the very low pressure area, a low temperature, trapped there on the coils of the condenser.

That is your introduction to Lyophilization and your introduction to the anatomy of a lyophilizer. I hope that you will join us for the short course workshop so that we can go into more detail.



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