Thermal Characterization as Part of an Empirical Process for Developing Optimized Formulations and Lyophilization Cycles

November 19, 2015

At the end of this webinar, you will be able to develop a well-defined process for taking an empirical approach to designing formulations and the lyophilization cycles used to dry them. By understanding and applying these principles, companies have a much greater chance of getting products approved by regulatory agencies than companies that employ a “trial and error” approach to formulation and lyophilization cycle design. 1 hour, 22 minutes.

Presenters: 
J. Jeff Schwegman, Ph.D., AB Technologies, Inc.
and Ruben NieblasMcCrone Microscopes & Accessories

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Transcript

Charles Zona (CZ):

Hello and welcome. My name is Charles Zona and I would like to thank everyone for attending today’s webinar. Our topic today is Thermal Characterization as Part of an Empirical Process for Developing Optimized Formulations and Lypholization Cycles. Our presenters today are Dr. Jeff Schwegman, the founder and CEO of AB Bio Technologies. Jeff specializes in formulation development, lyophilization cycles and thermal characterization studies including freeze-dry microscopy and DSC. He is available for speaking engagements and consulting services. We also have with us today, Ruben Nieblas of McCrone Microscopes and Accessories. For more than a decade Ruben has installed microscope systems all over the world and specializes in the installation and use of Linkam thermal equipment including what we’ll discuss in part today—Linkam’s FDCS196 freeze dry system.

Jeff and Ruben will also be teaching a three-day course on Lyophilization: Practical Applications Utilizing the Latest Equipment. The course takes place at Hooke College of Applied Sciences. Please visit the Hooke College website for more details about the course.

Both Jeff and Ruben will field questions from the audience immediately following today’s presentation.

Today’s webinar is being recorded and will be available on the McCrone Group website.

And now I’d like to hand the program over to Dr. Jeff Schwegman.

Dr. J. Jeff Schwegman (JS):

Like Chuck mentioned, today’s webinar is talking about thermal characterizations as part of a process that we use to develop optimized lyophilization cycles. I spent my entire career in contract development manufacturing, and still, it’s not unusual for a client to come to us and say that they’ve got a new product and they think it needs to be freeze dried, and, well, here’s the cycle we used on our product several years ago—just use that. That’s really not how things work. Each product is going to be a little bit different based on its thermal properties, and we have to design the cycle around that. So, the first thing we would need to do is to understand “What are the thermal properties of that product?” and that’s what we’re going to walk through today, and talk about a few case studies where we’ve had actual client products come in; getting into thermal characterization—some of the things that we found to not only optimize the cycle, but also optimize the formulation. You don’t want to shoot yourself in the foot designing a formulation that’s going to be very difficult, or next to impossible, to freeze dry down the road.

So the goal of lyo-development is sort of intuitive; it’s to design the fastest, most robust cycle that consumes the least amount of energy, so it’s running in the shortest time possible, essentially, and still getting the same quality product. That’s the goal.

The choice of values for the operating parameters have to be based on sound physical characterization of each formulation. This is something that within the past 20 years has really become commonplace for most companies doing lyophilization. It’s not just a trial and error approach where you put something in the freeze dryer, push a button, and hope you get something that looks good at the end; maybe as long as two weeks. You have to really characterize the formulation using good scientific principles to not only optimize the formulation cycle, but this is something that the Food and Drug Administration (FDA) is now expecting if you have a freeze dried product.

This slide is great, because it shows a couple of things.

Freeze drying, or primary drying, is sublimation work: converting the ice that we’ve created – we’ll talk more about how that’s created. We’re converting ice that we create from a solid to a vapor, and removing the vapor from the freeze dryer. It’s temperature dependent; exponentially dependent. When we look at this graph, it shows me that it’s a function of temperature.

So what we find is that by changing the temperature, we change the vapor pressure of ice, which essentially changes how quickly that ice changes into vapor. And it’s not a direct relationship; it’s actually an exponential relationship, as we can see here.

If we’re running our product temperature 10, 20°C below where we need to be, we’re substantially slowing down that process. This is what we’re going to try to understand today, is where is that magic temperature in here where we can actually run the product safely. If we run it too warm, we damage the product; if we run it too cold, we’re running it longer than we need to—just wasting time and energy.

This is a standard print-out from a freeze drying run. You see the freezing phase, and primary drying once we turn on the vacuum—again, we have to make sure the product is adequately frozen. Once we turn on the vacuum, we’re technically in primary drying. The goal of primary drying is to remove the ice. We get a phase separation, which we’ll talk about here in a little bit. And then secondary drying—once all the ice and the ice channels are gone, technically we’re in the secondary drying phase. This is where we’re removing, what we call, unfrozen water, residual water.

So in process development, what we’re going to be doing—and this is exactly what we do in our lab when a client sends samples to us—we characterize the thermal properties. We identify a couple of key factors: Is it crystalline? Is it amorphous? What are the critical temperatures that are associated with that? An then we start identifying the critical process variables around that, including the freezing rate, if we need to anneal (anneal a sample)—we’ll talk a little bit more about that, the time and primary temperature and pressure, same thing in secondary drying, as well.

Let’s talk about a few different phases here, and if you’ve done any reading in the literature of freeze drying, these should sound very familiar; they’ve probably popped up a little bit.

A eutectic mixture is an intimate mixture of two or more crystalline – now crystalline is a key – species that are in such close contact that they melt like a single, pure substance. One of the crystalline species might be an excipient, or an active ingredient; the other one is going to be ice crystals. We’ll talk about how these form, but it’s crystalline species, whether it’s an excipient or an active ingredient, and small, tiny ice crystals that melt like a single, pure crystalline substance.

Contrast that with the glassy phase. This is an amorphous species that forms a solid. There are no bonds formed here, and basically what we’re seeing is essentially molecules coming together. They get thicker and thicker, and colder and colder, until they finally form a solid. There’s basically a shift in viscosity that we’re experiencing with that.

Let’s talk about what’s going on here when samples freeze. We start off in solution. As we go down, we actually do a decrease in temperature. We cool the sample down. Somewhere below 0°C, we actually get two water molecules coming together, and if they can form a stable nucleus, they will go ahead and form a nucleus that will then propagate ice crystal growth. As we keep going down in temperature, basically, we grow the ice crystals and the ice channels—everything else is pushed around those ice channels into what we call the interstitial space, and then we get one of four different things that will happen.

The solute might crystallize. So we would say, “Okay, there’s still water in the interstitial space; it’s getting concentrated because we’re pulling the water out to grow the ice crystals, but there’s still some water associated there.” It’s going to go ahead and crystallize with the other components in there that are going to form crystals, and form that eutectic.

We also may see a stable glass. This is where the solution is getting cold, it’s pulling the water out to grow the ice and the ice channels—super-concentrating it. Finally, it gets so thick and viscous that it forms a solid. That’s what we call the glass. There are no bonds being formed, no lining up of the molecules; it’s just forming a solid that’s a shift in viscosity.

Then we talk about a metastable glass. This is something — this phase should have formed as stable eutectic, but for a number of different reasons, the molecules didn’t line up and form a eutectic. Instead, they just solidified in disarray. Now it’s not the most stable form; the stable form is the eutectic, so what’s going to happen is, if you freeze dry this, as the metastable glass, eventually it will go ahead and crystallize, and then it’s going to wreak havoc on your formulation. We want to make sure to get rid of that. That would be the process where we would talk about adding an annealing step actually acts to crystallize that metastable glass back to the stable eutectic.

Let me talk about a few key points here. That we’re going to, number one, identify what we have in the formulation. Is it a crystalline eutectic? Is it a glassy phase? Amorphous? I will tell you from experience a lot of the formulations we work with now, there is a combination of both, and we can actually see that in our testing techniques, so we’ll go through that. But what we’re looking at here, not only what is our formulation comprised of—crystalline, amorphous, both, metastable; and what are the critical temperatures that are associated with it.

If we look here again at the eutectic that formed, it’s got a temperature where, above that temperature, it’s not a solid, it’s a fluid. Same with the glass transition temperature Tg’ (Tg prime). Above Tg’, this phase is a fluid; it’s not a solid.

Now the problem is during primary drying, when we freeze the product, the ice that has formed in the ice channels acts as scaffolding, and when we go to primary drying, what we’re removing is that ice in the ice channels. If the interstitial space—where everything else is located around the ice crystals—is not a solid, this is where we would get collapse of product. We’ve got to be below the eutectic melting temperature; we’ve got to be below the glass transition temperature, or we’re going to get collapse of that product.

We verify that with freeze dry microscopy equipment. We run the thermal characters, the thermal analysis. What is it? What is the temperature that’s associated with it? Then we put a micro-drop on a microscope—a specialized microscope with a mini freeze dryer on it—and then we’ll actually watch it collapse as we go through different changes of temperature.

Why are these important? Well, as I mentioned, the eutectic, or glass transition temperatures for the most part represent the warmest temperatures we can take that product during primary drying. If we exceed that temperature, the interstitial space is a fluid. The ice is still intact. The ice won’t melt until we exceed 0°C, so the sample looks the same; we can’t physically see it, we can see it under the microscope but not on the other equipment that we look at. Above those temperatures, the interstitial space is a fluid. When we pull the ice out through primary drying, it can’t support its own weight, and this is where we get collapse of the product, which is not good.

Let’s talk a little bit about unfrozen water. Depending on the phase that forms in our sample, whether it be crystalline or amorphous, it’s going to trap water very differently. The crystalline phase, when it forms that eutectic, the molecules line up in a very precise pattern that they call the crystalline lattice, and unless we’re forming a hydrate, there’s really no room for a water molecule to get incorporated inside that crystalline lattice, so it gets kicked out to the surface. And these are much easier to lyophilize when we go to secondary drying. For a completely crystalline phase, at the end of primary drying, the product is already about 99.99% dry. For an amorphous phase, or a partially amorphous phase, we may trap—10% to 50% of the unfrozen water is still locked inside that glassy phase, and I think I have a little drawing here up a few slides that we can kind of dive into that. These two species dry very differently, both in primary and secondary drying, and how we design our cycles has to take that into consideration.

We talked about in primary drying, stay below the glass transition temperature Tg&prime or Te (Te = the eutectic melting temperature), or we’re going to get complete loss of that structure, and get a completely collapsed product. The moisture is going to be high, the reconstitution is going to be terrible, it’s going to look awful, and be completely unacceptable.

On the left is what we would call a crystalline phase. When we freeze, you see the ice in the ice channels, and then between the ice we have that eutectic mixture; we know it’s crystalline—there are tiny ice crystals in there, and tiny crystals of excipient or active ingredient. We like these nice wide ice channels, because they provide a conduit to get the water vapor out. The wider the ice channels, the easier it is to get that water vapor out. Now let’s contrast that with this glassy phase. We’ve still got the ice in the ice channels, but now this interstitial space is a glass, and water is actually embedded/locked inside that glass as unfrozen water, and it’s very difficult to get out. It basically has to diffuse through that very viscous solid to the surface of that glassy phase, and then volatilize and go out the ice channels down to the condensers.

In secondary drying—like I mentioned, it’s very different how we approach secondary drying, depending on what’s in our formulation. If it’s a crystalline formulation, we can be very aggressive in how we approach secondary drying, both in the ramp rate—maybe a little bit more in temperature, but we’ll talk a little bit more about that.

These critical temperatures represent the warmest temperature our product can go during primary drying without losing structure. I would never really start my primary drying at that temperature. Say our testing is at -20°C, the critical temperature, we’re going to start probably 5°C to 7°C below that because product temperature does tend to rise during primary drying, and we don’t want to exceed that critical temperature until the end of primary drying. You run the risk of either full collapse or partial collapse, cake shrinkage, things of that nature.

So, how do we do it? This is the big question. There are a couple of different ways. The first four bullet points here are what we would call the thermal analysis, and then we back that up with freeze dry microscopy, which gives us the complete picture of the thermal properties of our product. In our lab, we have the modulated DSC, it’s the gold standard anymore, but we’ll talk just a little bit about some of these other technologies, as some companies that have been around for a while may still have some of these, and utilize these as part of their development.

Let’s say a little bit about thermal analysis. For those of you who took physical chemistry, if you recall in thermodynamic, physical or chemical changes that occur with changes in temperature are generally accompanied by either an absorption or release of a small amount of heat. That’s what we’re picking up; that little, tiny amount of heat that’s given off, or absorbed, as these samples go through their transitions of melting, crystallizing, annealing; that’s what we’re picking up with this instrument; what we would call the thermal analysis.

Most common that we work with: differential scanning calorimetry (DSC), again, that’s the gold standard of what we have in our lab. Some people still use the differential thermal analysis (DTA), so we’ll say just a little bit about that.

Another technique that we’ll talk about is thermoelectric analysis (TEA). I think most freeze drying companies that make freeze dryers still offer this. It’s a very simple electrode that you slip down into a pair of electrodes that you slip down into a solution, and you’re measuring the resistance of the current flow between the two. It’s a very simple technology, and to be honest with you, it’s very limited in the information it can give you when you use it. So that’s why, again, if you stay current in the literature, it’s DSC that has been the gold standard for this.

For DTA, differential thermal analysis, we take a sample—now the sample that we’re going to use both for the freeze dry microscope, the DSC, or the DTA, it’s our liquid formulation that is formulated just like if you would take it and put it in a vial and put it in the freeze dryer. It has to have everything the same. We take the sample, and then we take a reference material, and then we cool the sample down. We’re taking it down to some sub-zero temperature. In our lab, we’re going down to about -90°C, and the reason we do that is—being a contract development organization, we see all kinds of samples coming to us with varying thermal properties. If we don’t go down enough, we may not see it. We’ll talk more about that range of temperature where we say “This is considered acceptable,” or “You may want to consider changing your formulation.”

So the DTA – the sample is cooled down to some temperature and frozen, and then we start warming the sample up. Basically, what we’re getting here is just a simple peak, and this is the temperature differential between the reference pan and the sample pan. The sample pan is going to go through a transition; it’s going to crystallize, it’s going to give off heat, it’s going to go through a eutectic melt, absorb heat. And this is measuring the thermocouples that are monitoring the sample pan and the reference pan, and it’s simply calculating the (delta) ΔT. So what this is telling me: oh, probably about at -30°C, -32°C, -33°C, we have a really strong eutectic melt here, so we have to keep the sample temperature below those temperatures.

This is an image of the DTA. Very simple technology, and again, you see the thermocouple measuring the temperature of the sample cup, and then also the reference cup with that ΔT. DTA has been used more frequently in the past; it appeared earlier in the market and the literature. It is a heck of a lot less expensive than DSC, but you don’t get the same information. It’s not going to be as sensitive as what we see with the DSC. The DSC technology has really come leaps and bounds in the past ten years. So many industries, other than what we do in the pharmaceutical industry, or the diagnostic industry are using these. There’s a lot of really good science and engineering that are in those DSC instruments.

Thermoelectric analysis: this is where we would take two electrodes, put them in the solution, and then what we end up seeing is a current that has passed; there are ions in solution, so they’re able to put a charge, or a current, between those electrodes when it’s in solution. What happens is, when we freeze the sample down, it locks those ions up, and they don’t carry the charge as well, so we see a big jump in resistance to the current. And then we warm up, and we go through a melt, and it’s able to carry the charge, and you see a big drop in the resistance.

I’m not going to go through the equipment. This is something that—you can buy these. I know that SP Scientific makes the Lyostar, and I think that Millrock, who has been working with us on the Hooke College course, I’m pretty sure they make a TEA unit electrode that can be plugged into their freeze dryer and used to get this data.

Log resistance is what we’re measuring. Resistance drops sharply after warming at the eutectic melting temperature because the charge carrying species are locked up; they can’t carry the charge, so we see a big increase in resistance when it’s frozen, and a big drop in resistance when it’s thawed.

This is just showing you a typical diagram. You can see at low temperatures, the resistance is quite high. As we increase temperature and it goes through its melting event, we see the resistance drop. What they’ve done here in the insert is taken the second derivative and get a real nice point that tells us, for this particular case, if we get much warmer than about -22°C we’re going to start to see this thing go through its eutectic melt.

I mentioned this technique is a little bit, you know, it’s useful for very simple systems, but if your formulation gets a little bit complex, it’s going to be a little bit misleading. We see a simple 5% mannitol in water. We know mannitol has a eutectic melt at -1°C, and that looks pretty good. We see that resistance drop once it gets a little warmer than -1°C. But again, the results become a little bit difficult to interpret as we put additional excipients in it, and you can see here. This is a combination of sodium chloride and mannitol. We see a transition for the sodium chloride; mannitol, it’s very difficult to interpret what would be the critical temperature.

This is simply changing the ration of sodium chloride to mannitol, and it’s all over the place. We really don’t know what’s going on with this.

It’s good for simple eutectics. If you have a one-component formulation, which there are many out there, it’s just the active ingredient dissolved in water and freeze dried, this might be good. It’s a very inexpensive to get some of this data. But a lot of the formulations that we’re developing today are pretty complex active ingredients, and they’re complex formulations, so this technique is sort of not going to be suitable for these complex formulations that we’re working with these days.

Let’s say a little bit about the DSC. In the DSC, the sample in the reference pan, just like before, are subjected to a carefully-controlled temperature program, Like DTA, we’re also cooling that sample down. In our lab, we have a refrigerated chiller that can go down to -90°C—it’s got a compressor system that will go down to -90°C. So we take them from room temperature, load the liquid in the sample pan, about 15-18 µL (microliters). The reference pan is an empty pan, cool them both down, and then warm them both up to look for the transitions.

The major difference between DSC and differential thermal analysis is in DSC, we’re not measuring change in temperature between the reference pan and the sample pan; we’re actually measuring heat flow. What the instrument is doing—it’s very carefully monitoring both the product temperature and the reference temperature, and it wants to keep them at the same temperature. As we’re cooling down, the sample is going to crystallize and give off a little heat. When the instrument senses that, it pulls heat away from the sample pan to keep them both at the same temperature. We’re actually calculating the amount of energy, or heat flow, that’s given or taken away from the pans as they go through these transitions. As we decrease the temperature, we’re monitoring the heat flow that is added to the pans.

The heat flow was additive sample; the heating rate is something we can control. Generally, I’m doing my heating and cooling at 10°C per minute. That does play a role in the sensitivity, and we’ll say a little bit more about that as we go. What we’ve got here, is, we can take the heat flow, divide it by the heating rate, and that gives us the heat capacity. This is one of the other things that really differentiates DSC from DTA, is we can now start to extract some of the thermodynamic variables. Heat capacity, if we integrate the heat capacity as a function of temperature, we can get the enthalpy for that complete reaction, and that’s great. Do we do it often in the industrial world? No, we did it a lot in grad school. If you’re making, you know, writing research papers, it may be something that you are very interested in doing.

This is the instrument in our lab, this is the TA Instruments’s DSC. Basically, we pop the lid off here. TA is a very innovative company, and they make these things with all the bells and whistles, so you can get autosamplers, and all this stuff—you kind of put your samples in the autosampler, push a button, and go home for the evening, and tomorrow or the next day, you come in and you have all of this great data that’s been generated. We don’t have that luxury here. But you pop the sample pan off, and there’s your sample and your reference pan; we just pop them in there and run our program. We can do this—this instrument is useful for the characterization of both liquid state, for finding, say, both Tg’ and the eutectic melt; we can also do it on the freeze dried solids. Now, why would that become important? Well, say for example, we have a formulation that’s mostly amorphous—and I mentioned to you that the amorphous phase traps water, and holds water pretty well. The problem is, that water will affect the temperature we can take the final product. Water is what we call a “plasticizer,” and the more water we have in there, the lower the temperature. We’re measuring the glass transition temperature in a dried state, we’d have to talk instead about Tg; the Tg’. There’s only one Tg’ in our formulation; when we first freeze it down, it’s got a fixed water content and a fixed temperature where it will go through its glass transition, from a liquid to a solid. When we’re in the dried state, there are many Tg values, and it’s all a function of how much moisture we pull out. When we keep driving off moisture, the Tg goes up. This instrument’s great for doing both.

This is one of the DSC sample pans here, and you can see it’s just very, very tiny. We use a little more than 10 µL; I think we’re now using about 15 µL. It will affect the sensitivity—the amount of sample that you use; there is a certain limit, obviously, due to the pretty small size of the pan.

The power compensation principle—this is all based on heat flow. We’re taking away power; we’re giving power to the system to keep the sample temperature the same as the reference pan temperature, and then we’re keeping track of how much energy we use, heat flow, to do that.

When using DSC for conducting a thermal analysis study on a lyophilization cycle, we generate two curves. One would be a cooling curve, so we measure the curve. We start at room temperature, go down to -90°C, we do a slight hold at -90°C to let everything kind of equilibrate. Then there’s one curve—we see the freezing event. Then when we warm up, we get a warming curve. We don’t necessarily use the cooling curve for anything other than looking at it—does it freeze at a reasonable temperature. I’ve worked with products that didn’t even freeze until you get them down to about -50°C, and in that case, yeah, we have a problem. You’re not going to be able to get it frozen, likely, in a freeze dryer. Maybe you could squeeze those temperatures out of a development scale dryer, but with commercial scale dryers, it’s going to be very difficult to get that low and maintain a temperature that low.

We see here a typical DSC scan. The bottom is the cooling curve, so we start at room temperature, and boom! We see that big peak. That’s crystallization of ice. Notice the temperature. Everybody knows that ice melts at 0°C, and that’s true. Ice does not crystallize at 0°C. Specifically, in these cases, we’re working with formulations that contain additives, excipients, that contribute to what we call freezing point depression. There’s also something called supercooling. Generally, for most of the formulations we work with, we’re going to get crystallization, usually between -10°C and -20°C. I’m looking at this to make sure the temperature is not down in the -50°C region. The top curve is the warming curve. What we’re going to look at is, let’s go and take a look at some of the warming curves here.
So these are the warming curves. We’ve cut the cooling curve off; we’ve looked at them, and gotten rid of them. What we are seeing here is a classic example of a eutectic melt. We see a very sharp peak, it’s symmetrical, it’s very high-energy, so we would never miss that when we’re doing a thermal characterization.

When we talk about the temperature that’s going to be used for the onset, the computer software draws a tangent to the leading edge of the baseline, and a tangent to the leading edge of the peak, and the midpoint where those intersect is what we technically call the eutectic melting temperature.

The glassy phase, the glass transition here, is what we call, kind of, the S-curve. Basically, the computer draws a tangent to the leading baseline, and a tangent to the trailing baseline. The midpoint where they intersect is what we classically represent as the glass transition temperature. This on top is what we call a metastable glassy phase. We know that, because what we see is we’re warming up here. You see the small glass transition right here. This is enthalpy recovery, which is a little bit out of the scope of this webinar, and then, boom! We see this big exotherm—that’s crystallization. So, it’s a solid down here at about this temperature, -30°C, we warm it up above the glass transition, it goes through this fluid state, and then, very quickly, the molecules line up and crystallize. This is a classic example, so when we see this, we know that we have a metastable system, and we’re going to have to add an annealing step to the process.

What if we notice that we can’t detect the glass transition? This is entirely possible. If we look here at the glass transition, you’ll notice the noise in the baseline, and that’s because we’ve had to magnify this extremely large to here even to put it into perspective. It’s a very low-energy transition. If you think about a eutectic melt, it’s ripping bonds apart, crystals are being torn apart; in the glass transition, that’s not the case. It’s just a shift in viscosity. Unless we go looking for it, we’re not going to see it.

There are a couple of things we can do. Increase the heating rate; we generally do our first run at 10°C per minute, we can go as high as—the system is capable of going very, very quickly, maybe 20°C of 30°C per minute on the warming curve. We can also add a little bit more sample within the limits of the sample pan.

This would be a good, classic example of running the system. And taking this for face value, I might say hey, here’s a big eutectic, I would say the critical temperature is about -4°C, -5°C; so why don’t we run our process at -10°C? We should be able to get a good product. What we find in the back baseline, once we go looking for it, is this low-temperature glass transition. What that means now, instead of freeze drying, primary drying, at -10°C, we’re primary drying down at -46°C, -47°C, to keep it from going through a collapsing event. The problem is, if we could freeze dry this product at -10°C, we could probably freeze dry everything in 12 hours. Now we’re freeze drying down at -46°C, you’re probably looking at four or five days. As I mentioned, the colder we keep it, it lowers that vapor pressure of ice, hence, it lowers the sublimation rate, and it just exponentially slows down that process. When I would see something like this, I would tell that client that they just have to reformulate.

It’s important to know early on; do studies on your product to see if there’s any potential that the product might need to be freeze dried. We could do some very early accelerated stability studies. Does this stuff degrade through hydrolosis in the liquid state? Because, what you don’t want to do is get to the—ready to manufacture, and you put your formulation together without someone who understands lyophilization, and they find it’s very difficult or impossible to freeze dry. This is what you don’t want. My first response to a client would be, well, there’s something in your formulation that’s causing this low-temperature glass transition to be present in your product. Can you pull that out? Or can you pull some of it out? Sometimes that’s yes, sometimes that’s no, and there are some other strategies that we can do, which we’ll talk about, to improve that product or that process.

What if we have a eutectic and ice, but only see one peak? This is not an uncommon occurrence, and we see this quite often with mannitol. Remember, on DSC, we’re running this at a liquid; we’re freezing a liquid and then re-melting the liquid, so there is still ice present. We see the melting of ice at 0°C; it’s a huge peak, because ice or water is the substance mostly what’s in there, and mannitol has a eutectic melt at -1°C. There going to overlap. We’re not going to see them. Is that important? Not necessarily; we’ve done a few experiments where we’ve resolved those, but the temperature where that event starts is not going to change; you’re just resolving the two. It’s not going to give you a lot of information, unless you’re doing research. The way we can do that is by slowing the heating rate down. Increasing the heating rate increases sensitivity; decreasing the heating rate increases the resolution of the instrument.

This is just an example I’ve thrown in of glycine. Glycine has several different polymorphs. When we ramp it at 10°C per minute, they all sort of melt the same; similar times, overlapping times. However, we slow that heating rate down, and we start to be able to resolve those different melting events of the different polymorphs of glycine.

My DSC rule of thumb is if resolution is the issue, decrease or slow down your heating rate. If sensitivity is your issue, you can warm up or increase that heating rate, or try increasing your sample size. It’s as easy as that.

I’m not going to go through the techniques in detail in the essence of time, but you can refer to this. This is how we do the liquid samples; 10 µL, now I’m using more like 15 µL, but they’re hermetically sealed. I didn’t mention that before. The reason we do that is actually sealed, crimped against the outside environment. The reason we do that is to prevent evaporation. Sometimes we’ll put these things in the pans and they’ll sit round a little bit before they’re tested, and we don’t want evaporation to occur. So by hermetically sealing that pan, we’re eliminating the evaporation and change of the thermal properties.

We can also do this in solids. As I mentioned before, this becomes important if we want to know, well, what temperature can we place our freeze dried sample at and not have it collapse after the fact. I’ve seen it happen. They’ve prepared a freeze dried sample at the previous company, and they shipped it out to—I think it was Arizona—on a non-temperature-controlled truck, and when it got there, they were all collapsed. Number 1, we shouldn’t be shipping on a non-temperature-controlled truck; and number 2, we need to actually increase the secondary drying time to pull out more residual moisture. These things were even collapsing on stability; accelerated stability. By pulling out additional moisture, we raise that glass transition to where we can actually store it at these higher temperatures without losing structure. That’s the temperature; we can find Tg using our DSC, as well.

Let’s talk a little about another technique we use in conjunction with DSC, and that would be the freeze dry microscopy system that’s sold exclusively in the United States through McCrone Microscopes & Accessories. Basically, it’s a direct examination of freezing and freeze drying by a special microscope and thermal stage that we put on the microscope. We do this as well. We do the DSC first, we do the microscope. These two pieces of equipment and the data we get from them really complement each other. They’re giving a little bit different pieces of information that we use to get the complete thermal profile from our sample.

I have to apologize; this is an older system we have in our lab. Ruben from McCrone has an image of the latest and greatest, and they’ve modified a few things, but basically, it’s the same process. It’s a polarized light microscope, There’s our little freeze drying stage which I’ve got a picture of that in more detail, We have a little vacuum gauge there, it’s cooled with liquid nitrogen, so there’s a dewar, there’s our vacuum pump, and these are the controllers and pumps for the liquid nitrogen.
Some of the necessary equipment…we’ll talk a little bit about some of these that makes them different from a normal polarized microscope. It’s one of these things that I tell you, I have seen some people try to build these things themselves, and it’s never worked out. McCrone sells these as a complete package; they’re the only dealer in the United States, so if you’re interested in purchasing a unit, talk to them about that. You don’t want to try to do this on your own.

We’re going to walk through some of the differences here that we may see between this and a regular polarized light microscope. This is what we call a first order red compensator. You’ll notice, when we get to look at some of the images, the images are colored. This is something that’s used to do that. It’s essentially a 530 nm (nanometer) filter that we put in line, and it allows us to see different colors once the light’s been polarized past the sample and then goes to the analyzer.

I talked about the first order red compensator, I’m not going to say anything else about that. The other thing that’s really interesting is the long working distance objectives. For those of you who took microlab, you put your sample on the stage, and get your objective lens very, very close to it so you can focus. A little bit different here. You’ll notice down below I’ve got a partial picture of the little freeze dry microscopy stage, and the sample sits right in the middle of that. We couldn’t get the objectives close enough to focus on them; the focal length is beyond what we can do with the regular objective lenses. These are specialized objective lenses with a longer working distance, or focal length, if you will, so we can actually focus down into this little mini freeze dryer.

Again, a vacuum pump. Linkam is the company that makes the freeze drying stage. They’re very innovative. They’re talking to the guys in universities using this, and in industry…what do you like, what don’t you like. One of the things everybody wanted was a vacuum control valve that was controlled by the computer. Basically, you tell the software, I want to run this process at 100 millitorr; it automatically opens, there’s a needle valve in there that opens and closes this needle valve to achieve the correct pressure that you set.

This is a special Linkam stage. It’s a really innovative device. It essentially clamps on to the stage of the microscope. The lines are in the back here for the inlet and outlet for the liquid nitrogen. The lines here are the vacuum: this one holds the Pirani gauge, and this one holds the line to the vacuum pump. What you’ll notice, these two little knobs on the side, those allow us to manipulate the sample in the X-Y plane. It’s a really innovative technique, too, because, the way we used to do it, the sample sits here, and you can see that it’s a 1 mm diameter window. If the sample is sitting there freeze drying, and the sublimation front that we’re trying to watch and follow passes that hole, we can’t see it anymore. They’re really innovative. They’ve got this little device – they call it a lollipop – but it’s connected to that X-Y manipulator, and allows us to move the sample through while it’s freeze drying and follow that sublimation front. Really neat. This here at the bottom is the guts of the thing again. Really innovative piece of equipment.

I’m not going to say too much in regard to polarized light microscopy theory, but it’s possible to tell if our sample is crystalline or partially crystalline due to what we call the birefringence of certain types of crystals that we see called anisotropic crystals. What we mean by anisotropic, these are different crystal forms, or amorphous forms, that we might be able to see. The first one here is what’s called an isotropic crystal. What that means, is, it’s just, this is sodium chloride, it’s a sodium atom, chloride atom, sodium chloride. It doesn’t matter how you’re looking at this thing, if you turn it on its side, turn it upside-down, you’re still going to see that pattern of sodium and chloride.

It’s not so, in this case we’re looking at calcite, it’s what we call an anisotropic crystal. It has this crystallographically distinct axis, which means, simply, depending on how you turn that crystal, you’re going to see a different end of the molecule. These bend light extremely…very differently when we pass polarized light through it. Again, it’s amorphous; it’s not going to do anything. We’ll show you an example under the microscope of what we might see when we look at an amorphous component, or a component that’s not crystallized.

So I mentioned that anisotropic crystals have crystallographically distinct axes, and interact with light by a mechanism depending on the orientation of the crystal.

The technique—we’ll just kind of run through this; it’s a pretty simple thing. Again, with a lot of experimentation that has gone on, we’ve really worked out a really good technique both in the equipment that we use, the supplies, and also, just some of the tricks that we’ve used. We’re using quartz windows instead of thin glass—better heat transfer. We’re putting a small, tiny drop of silicon oil on the base of the stage, and then the bottom quartz window goes on that, and that does a couple of things. It improves the heat transfer from the block to our sample, and provides lubricity as well, so we can easily move the sample around.

We open the top, put the lollipop in, drop the silicone oil, quartz window—I use 5 µL of sample—put another smaller window on top, seal the chamber, and start my cooldown to my freezing temperature. Set the vacuum. Well, we’re always collecting images in this state as well. We always include the images in the reports that we submit to clients. So we’ll freeze it and collect an image, then we’ll turn on the vacuum. We’re going to start freeze drying this at a safe temperature; meaning the DSC said, for example, you’ve got a glass transition at -20°C, I’m probably going to start freeze drying at -30°C. The reason I do that is I want to collect an image of a sample that is not collapsed. It’s got an intact dried layer, we can see the sublimation front proceeding nicely; I’ll collect an image of that. Then I’m going to start raising the temperature, and I don’t want to force this into a state of collapse, because then I record that temperature. We’re actually recording two temperatures when we do this for clients; we say we’re going to record what’s called the onset of collapse: it’s the temperature when we start to see tiny little pores just begin to open up; and then full collapse when the entire region falls apart.

Experimental details, again, this is something that if you buy an instrument, Ruben, their rep, will come out and set it up for you, give you all the training you need, and these are the things that he will tell you the details about the supplies we use, the techniques that we use to plate the sample and dry the sample.

Let’s look at some images here. This would be what we call a eutectic melt. I will tell you that this image has been modified, in the fact that we specifically manipulated it to get really large ice crystals. Generally, this is not going to be what you are going to see under the microscope when the sample freezes, but we did this to show you the difference. The squiggly lines here, that’s pure ice in the ice channels. Everything in between it is our interstitial space, in which contains the active ingredient, all the excipients, salts, surfactants; you name it. Everything else in our formulation is crammed between those ice crystals. It’s a little bit difficult to see in these, but there’s little colors, there’s a little color associated with that interstitial space, and that’s due to the birefringence of the crystals. The magenta, or pink, color here, that’s due to that red compensator. If there are no anisotropic crystals present, the sample just looks pink due to that filter. At this temperature here, the ice is frozen, the interstitial space is a solid, so we’re below the eutectic melting temperature for now. We raise the temperature, and we get this second image, and you’ll notice the interstitial space—there’s no longer color associated with it; it’s a fluid. We’ve exceeded the eutectic melting temperature, the interstitial space is fluidized; but notice, we still have ice in the ice channels. We haven’t exceeded 0°C and melted the ice. Really interesting images there.

This is an image of a partially crystalline with an amorphous component to it, frozen at -45°C. We turn the vacuum on, and we’re going to freeze dry at a conservative temperature, below where we saw the glass transition. We see here the frozen layer is intact, you’ll notice the differences in color. It’s all uniform, what we see in the frozen layer. You’re just seeing different ends of the crystals. So, in the blue region, we saw the left side of the crystal; and this is, again, to our field of view, what we can see under the microscope. When it was orange, you were seeing the bottom of the crystal, so whatever orientation of this thing when it solidified, it’s bending light differently. So what we have is the intact dried layer. You can see the sublimation front; it’s a razor-thin line between the frozen layer and the dried layer, and it is intact. Then we start warming the sample up. There’s another image I need to put in here, but basically, if you look back here, in the region here we see these tiny, little pores open up. Those tiny, little pores—that’s what we would report as being the onset of the collapsing event. As we get warmer, we exceed the glass transition temperature; that’s where we see full collapse. And this is where you’re going to see the collapse, right at the sublimation front. It tells me here that we need to be freeze drying colder than -36°C or else we’re going to have a problem with this product.

What have we learned from this? Basically, the thermal analysis studies, combining both the DSC and freeze dry microscopy have given us several very important key pieces of information, not only in designing our formulation, but also in designing our lyophilization cycle around it. It tells us is the formulation amorphous? Is it crystalline? Is it a mixed system that’s partially crystalline? Is it metastable? We can also do annealing studies on the DSC and the microscope to understand what the optimal conditions are for annealing our product. It tells us the critical temperature that is associated with it. What is it – crystalline, amorphous, metastable; what are the temperatures associated with it, and also tells us what the conditions are if we need to anneal the product.

What this allows us to do is take a scientific approach to freeze drying. This is not a trial and error process anymore; we don’t just put things in the freeze dryer, push a button, and hope that in a week or two, we get something that looks good. This is something that’s specifically scientifically studied, and the cycle is built around these thermal properties. In the old days when we were doing freeze drying, it might take 15-20 runs in a development scale dryer to get something that worked, and it probably wasn’t even optimized. With this information, so I do the DSC, I do the freeze dry microscopy, I can have an optimized cycle done in 3-4 runs. It’s a huge time-saver. The FDA is going to ask you about your thermal properties, what’s your glass transition temperature, so you need to do it.

Let’s run through a couple cases here, and these are always interesting. We get clients coming from every phase of drug development; it’s honestly very rare that we’ll get a client that comes to us and says, “We’ve got a new product. It’s showing some signs that it might not be stable in water. We want to work with you to help us design a formulation in case we have to go that route.” Normally it’s the guys that put something together that they try to manufacture, and they can’t, or they have problems. That’s generally what happens.

For Client #1, this was a diagnostic company and they wanted to convert their liquid product to a more stable freeze dried version; however, this stuff totally, completely collapsed. I don’t mean to harp on the diagnostic people, and I do apologize if there’s any in the audience, but generally, these are the worst formulations for freeze drying. I’ve worked with several different companies, and they send me their products for thermal analysis/thermal characterization, and it’s just god-awful. It’s impossible to freeze dry. So how do we fix this? What can we do to fix it?

Basically what we did…this was the DSC that we ran. I didn’t even see a curve. I only ran this down to -65°C; I should have gone lower, but I don’t even see a clear glass transition; it’s telling me it’s probably lower than -65°C, which is terrible. So then what we start doing…I’m sorry…then we ran the microscope, and again, just total collapse. This is down at -45°C, we’re freeze drying at -45°C and we’re getting absolute, total collapse of the product. I talked to the company, “Hey, can you pull…what’s in your formulation, there are certain things you can pull out,” and they were able to compromise a little bit and pull out a few things that looked bad, that I thought were going to behave badly, and they reduced some of the things that they could, and it still wasn’t enough. So we look at something called glass transition temperature modifiers, and these are great. Generally, amorphous phases will come together and form a continuous phase. It’s like blending them together when we freeze the solids down, and that’s great. The glass transition temperature of that resulting solid that, of the combined amorphous phases, that transition temperature is also a combination of the different temperatures.

Now, we’ve got their product with a glass transition temperature that we can’t even read, and now we’re blending in some other things that we like, where we know what the glass transition temperature is, what these are like, in this particular case we looked at dextran 40. We’ve also worked with gelatin in the past; hydroxyethyl starch, but dextran is a favorite. We just start blending in. This is formulation development, this isn’t lyo development, and I’m standing there with my DSC running, making these changes, and shooting it—boom! What addition that changed that formulation; how has that affected the thermal properties? This was
22 mg/mL of dextran blended into that first client’s product, and you can see that now we’re getting a glass transition temperature, in this case, -38°C. It’s not great, but at least we can see it. Now we just start doing higher concentrations as we go, that’s 22, this is 40—now we’re up to -29°C; we go to 60, now we’re up to -25°C. This is a great number. These numbers are well within what we can freeze dry and get a beautiful product; freeze dry it in a relatively short amount of time. We go up to 80, and we get -23°C; that’s fantastic.
This is just a summary showing, in this case, blending in higher amounts of dextran, which has an overall by itself in water has a glass transition temperature of -9°C, we’re really able to raise that critical temperature of that product where we couldn’t even freeze dry it previously, and now we’re able to freeze dry it at relatively warm temperatures. It’s going to freeze dry very well.

The product made beautiful cakes—dextran, it makes good cakes. It’s drying very quickly; I think this was just a couple of days in the freeze dryer. They put this stuff on stability, and it looked excellent; they’re getting good titer—this was an attenuated live vaccine, I believe; modified live. So they now have this stuff on accelerated stability. The only problem we’re having a little bit of reconstitution time issues; while dextran is great at modifying the glass transition temperature, it does tend to get a little bit gummy at the higher concentrations, so we had to maybe cut that back a little bit.

Client Product #2: This was an injectable drug product. They developed an injectable drug and this lyo cycle in the development lab scale dryer. These dryers are more efficient, both in heating and cooling rates, with low temperatures they can go to. The small-scale batches they were making showed good stability when they were looking at early clinical trials and stability studies. However, scaling up became a real issue. They were noticing in the larger dryers they were getting partial failure of the products. Some of the vials were collapsing. When we get a partial failure, the question that we have to ask is where was the location of those vials where the collapse was occurring. We noted that. In this particular case, they did note that…well, this was the glass transition temperature. It’s not bad, it’s -34°C; we could work with that. The problem was they didn’t want to change the formulation. So what I said was, well, your product (this is where we talk about issues with scale up), the product was running a little bit warmer than the production dryer, and the vials on the edge experienced something that we call edge effect. They see a slightly warmer environment due to lulls in the freeze dryer, and they were warming the samples just very close or over the glass transition temperature, and they were collapsing.

To summarize everything, both the DSC and freeze dry microscopy can, and should, play a major role in not only your formulation development, but also in the lyo cycle development. The FDA is going to ask for it, it’s just going to make your life so much easier in both the formulation development and the lyo cycle development.

The goal is to freeze dry these things as fast as we can, but we have to have good stability, there has to be all of the quality specifications laid out previously.

This is my contact info. Feel free if you have any questions. We’re going to turn it over to Ruben, and then we’ll open up the floor for questions; you may have questions now. Send me an email or give me a call if there are any additional questions outside of this that you might have.

CZ: Go ahead, Ruben.

Ruben Nieblas (RN):

Hello, everyone. Jeff, can you see my screen? (JS: Yes, I can.) Great. Well hello, everyone. My name is Ruben Nieblas, and I’m with McCrone Microscopes & Accessories, and I have a very short presentation here on the Linkam freeze drying FDCS196 system. Just a very quick introduction here so you know the equipment that is used.

So as Jeff had mentioned, how do we characterize our formulations? The two pieces of equipment that we use are, of course, the DSC and the freeze dry microscope. The information the DSC gives us is eutectic temperatures, and glass transition temperatures, the information from the freeze drying microscope gives us our collapse temperatures. As Jeff iterated, what we’re trying to do is optimize our cycle development, so for every 1° we can freeze dry warmer, we exponentially increase the rate of drying. Therefore, the glass transition does not always equal the collapse temperature. You can have anywhere between a 5-15° difference between what your glass transition temperature and what’s your true collapse temperature, and the only way to really find that is by using the freeze drying microscope.

Thermal analysis studies tell us if the system is amorphous, crystalline, or partially crystalline, tells us our critical temperatures, and it also tells us if we need to anneal the system and approximately what those conditions are.

So here is the actual Linkam freeze drying microscope system. I’m going to go from left to right and just go over the components that are on here. First, with any freeze drying system, we need a vacuum pump for the sublimation. This is a system that is provided with a BOC Edwards 1.5 Pump. You’ll notice on top of the pump there’s like a silver cylinder. That is actually the NV-196 motorized valve control. With the software, we can actually regulate the pressure inside of the stage itself, so you can mimic exactly what’s going on in your pilot or production freeze dryers.

The system is run with liquid nitrogen, so the system comes with a two-liter dewar that is filled with liquid nitrogen. The system with liquid nitrogen will last you anywhere between 4-6 hours, depending on how low you’re going, the rates, limits, and so on and so forth.

Next, we have the actual stage itself, the Linkam freeze drying stage on a polarized light microscope. The reason we have it on a polarized light microscope is for qualitative, not quantitative, reasons. We don’t care about the birefringence of the materials that we’re looking at, but what we’re trying to do is, we’re trying to get the best image possible of that freeze drying front as it’s moving along, so that we can determine when that collapse temperature is, on to it. So by using the polarized light accessories on the microscope, we get the type of imaging, and lighting, and contrast that we use to better determine where that onset of the collapse is. The systems are run…Linkam has designed their system to run with the Q-Imaging line of cameras, so that is also provided.

Next we have the little red box on the port on the right-hand side of the stage. That is your Pirani gauge. Using the Pirani gauge, we know what the pressure inside of the stage is, and that is the readout that we use to control using the NV-196: the pressure inside of the stage.

Next you’ll see that there are two boxes; the bottom box is your T95, or your temperature controller. The top box is you LNP95, or your liquid nitrogen pump. Those are the two items that are basically the brains of the system to run the system. The way that you can think about the system actually working, it’s like a tug-of-war. You have a platinum resistor inside on the block itself of the stage, which creates heat through electrical current run by the T95. You also have channels in the block, where the liquid nitrogen is flowing through. So therefore, when you tell the system to go 10° per minute, 5° per minute, let’s say down to -40°C, it knows how much electrical current to run through the platinum resistor, and how much flow of liquid nitrogen needs to go through the block. So you maintain the rate that you told it to. The system can go as slow as .103 per minute, or as fast as 150° per minute. The temperature can go down to -196°C and as high as 125°C.

Lastly, the system is run via a computer. If needed, we can always send you the specifications for the computer.

We will be holding a course, Lyophilization: Practical Applications Utilizing Latest Equipment here at the Hooke College of Applied Sciences in Westmont, November 3rd through the 5th.

If you’d like to learn more about the Linkam freeze drying system, or if you have any questions about the system, you’re more than welcome to contact me here at McCrone.

With that, if anyone doesn’t have any questions here, I’ll go ahead and transfer it over to Chuck, and he can go over some of the questions that have been posted.

CZ: Thanks, Ruben—thanks Jeff. That’s some interesting stuff there. If you have any questions, just go ahead and type them into the question box. And, take a look at some of these questions coming in here…

“What is the difference between Tg and Tg’?”

JS: Okay, that’s a good question. They both represent amorphous phases. Tg’ is when we put our amorphous phases in solution, and we freeze them down. When they freeze, they form…there’s a fixed water content that gets trapped inside the amorphous phase, and a fixed temperature where that amorphous phase will go through its glass transition. There’s only one Tg’ in your formulations called the glass transition temperature of the maximally frozen concentrate. When we start ramping up, or pulling moisture out, secondary drying, that temperature we can take that amorphous phase goes up. Water acts as a plasticizer, so there’s one Tg’, but as more water comes out, there’s many Tg values of that amorphous phase, because we’re raising the Tg hopefully above room temperature when we pull it out of the freeze dryer.

CZ: Okay, great. “What is the general variability in collapsed temperature values in replicates of freeze drying microscopy runs?”

JS: There will be a little bit of variability; it shouldn’t be much. I would say, in our experience, probably, maybe 1-2°C. Unlike the DSC, where there’s a hard number that’s generated when the computer calculates the Tg’, this is a visual examination, so we’re recording images. I might say, well, that looks to me like the onset of collapse, and somebody else might say, no,
I think it started over here, maybe a degree or two colder. We don’t see anything more than a degree or
two of variability.

CZ: “In lyophilized polypeptide antibiotics, we often face a lot of vial breakage when developing the lyophilization cycles. What would be the best approach to resolve that?”

JS: We’ve seen that quite a bit, too. There are a couple of rules that we try to stick by with freeze dried products. Number one, we don’t like to fill the vials more than half full. If you’ve got 10 mL vials, you don’t want to be trying to freeze dry 10 mL of product. The expansion is just too great. We would say if you want to run 10 mL, go to a 20 mL vial. The other thing would be, if there’s mannitol in the formulation, mannitol is very characteristic, it has a relatively large expansion coefficient. It will shatter vials at higher concentrations, so try to keep the mannitol below 3%. And then even the total solids of your product—if you’re making it super, super concentrated, you might be shattering vials; you may try to reduce that a little bit. Maybe reduce the concentration and just up your fill volumes. You’re still getting the same dose, it’s just the total solids per mL is not ridiculously high.

CZ: “At what point during the shelf temperature ramp of primary drying do you feel comfortable with the product temperature being above Tg’? Is there a general rule of thumb?”

JS: Well, when we’re doing the cycle development, we want to keep that product temperature below Tg’. Now, product temperature will rise during the primary drying process. My rule of thumb for that is, say for example, we do the thermal characterization, we get a clear collapse, glass transition temperature of -20°C, I’m going to start primary product temperature drying at about -25°C to -27°C. This is where you’d have to kind of play with your freeze dryer and do a little bit of development work to find out what that shelf temperature would be, and chamber pressure would be, to keep the product temperature at -25°C in our example.
CZ: “How did you determine the edge effect for the last product, product temperature measurements?” I think Larry [the webinar viewer who posed the question] here has…in that, he clarifies that in a production-sized machine?

JS: The edge effect…it’s…generally, we do it in development, but thermal couple probes throughout the dryer. Generally, if you’re going to see a collapsed vial in a large dryer when we scale up, it’s going to be near the door, near the wall; and in some case, we can actually go in and probe those thermal couples as well. Generally, what we’ll find is that in the dryer, the edge effect is a little bit greater than in our small development dryer. We can hopefully measure that with a little thermal couple, and what we’re going to have to say, well, we’re going to have to run the shelf temperature maybe 3° to 4° cooler when we scale up, but we also may have to run that cycle a little bit longer. So, we’re keeping the edge vials under the glass transition temperature, but it’s also cooling the center vials, and they’re going to dry slower than what we did in development. Again, this is all part of a good scale up study.

CZ: It looks like we have a couple more questions here. “What is the best heating rate in freeze dry microscopy to observe collapse temperature?”

JS: Well, it might be a little bit of preference. I mean, we typically ramp through the transition at about 5°C per minute. I don’t know if…Ruben…if you have a preference on that? You do quite a bit, as well, but we generally will use 5°C.

RN: Yes, about 3°C to 5°C per minute is usually what we run it at. You don’t want to go to fast, like 10°C per minute, because then, all of a sudden what you think would be is the actual collapse temperature, because you ran it so fast coming through, you’re not really getting the true collapse, just because of the rate of the heating coming through, especially at 10°C per minute. You might be overshooting that temperature, because you still have to give it actual time for the effect to happen. So usually, I’ll run it at about 3°C to 5°C per minute to get the true collapse temperature.

CZ: “How do you determine the annealing temperature?”

JS: The annealing temperature we determine from the DSC scan, so that slide I showed with a metastable glass…so we see the glass transition, boom, we see crystallization. What we didn’t see on that slide is, if we would’ve gone warmer, we’d see this giant ice melt curve. We don’t want to get into that. The temperature that we choose is based on after the glass transition, after the crystallization event is done, but before we get into the ice melt. That’s really the sweet spot for annealing.

CZ: Looks like our last question here: “What are the factors that need to be considered from lab scale to scale up process?

JS: That’s the million dollar question. There are so many variables that we have to look at. I’ve got a whole list of questions that I’m asking the group that knows the larger piece of equipment. There are differences in large freeze dryers’ design, there are different ways we monitor and control temperature; we can essentially scale up the process in a freeze dryer, we just need to make sure we understand the differences. Generally, the biggest factor we see, that causes problems, is product temperature is not the same. There is a whole list of things we want to go through and make sure we understand even before we design the cycle. I don’t want to design something that can’t be, you know, run in the bigger equipment. We’re asking the guys who run the larger equipment what are the maximum heating and cooling rates, what is the lowest shelf temperature achievable, the lowest condenser temperature, all these questions. You just really have to know the dryer scale, and know its limitations.

CZ: It looks like we had one more question come in, Jeff. “For secondary drying, what chamber pressure is typical: higher, or lower?”

JS: Well, the old way they used to do it was, at the end of primary drying, they’d go into secondary, they’d drop the vacuum as low as it could go. We don’t do that anymore. Studies have shown that it makes no difference whatsoever. And, studies have shown that if you go to an ultra-low or an ultra-high vacuum, you enhance the oil backstreaming from the pump getting into the freeze dryer. You do run the risk of getting some oil vapor mist into your product. We determine the vacuum level in development, and run it for both primary and secondary drying. We don’t change it.

CZ: Okay, great. Well I think that’s it for the questions.

Conclusion

I’d like to thank everybody for attending, and thank Ruben and Jeff for putting on a great, interesting webinar today.

We’re going to be having another webinar coming up at the end of the month, so be sure to join us on October 29th when our presenter Elaine Schumacher will discuss the Identification of Glass Delamination Products Using Transmission Electron Microscopy, and that will take place again at 1:00 p.m. We hope to see you there.

If you have any other follow-up questions, you can contact Jeff and Ruben through their contact information, and thanks again for attending today.

CONTACT A FREEZE DRY EXPERT

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