[Freeze Drying Microscopy Pdf Download
Tilo Chopin
FDM works somewhat like a miniature freeze-dryer. The sample is frozen inside a small microscope chamber by using liquid nitrogen. While applying a vacuum, the temperature is gradually increased and the sample dries. During this temperature ramp, the sublimation front is observed and recorded through a microscope. Changes in the dried layer can be used to determine the state of the system (amorphous or crystalline) and the collapse temperature of the sample.
FDM is mainly employed during lyophilization process development and formulation development, where it is combined with differential scanning calorimetry (DSC) to determine optimal freeze-drying conditions.
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Optical coherence tomography freeze-drying microscopy (OCT-FDM) is a novel technique that allows the three-dimensional imaging of a drug product during the entire lyophilization process. OCT-FDM consists of a single-vial freeze dryer (SVFD) affixed with an optical coherence tomography (OCT) imaging system. Unlike the conventional techniques, such as modulated differential scanning calorimetry (mDSC) and light transmission freeze-drying microscopy, used for predicting the product collapse temperature (Tc), the OCT-FDM approach seeks to mimic the actual product and process conditions during the lyophilization process. However, there is limited understanding on the application of this emerging technique to the design of the lyophilization process. In this study, we investigated the suitability of OCT-FDM technique in designing a lyophilization process. Moreover, we compared the product quality attributes of the resulting lyophilized product manufactured using Tc, a critical process control parameter, as determined by OCT-FDM versus as estimated by mDSC. OCT-FDM analysis revealed the absence of collapse even for the low protein concentration (5 mg/ml) and low solid content formulation (1%w/v) studied. This was confirmed by lab scale lyophilization. In addition, lyophilization cycles designed using Tc values obtained from OCT-FDM were more efficient with higher sublimation rate and mass flux than the conventional cycles, since drying was conducted at higher shelf temperature. Finally, the quality attributes of the products lyophilized using Tc determined by OCT-FDM and mDSC were similar, and product shrinkage and cracks were observed in all the batches of freeze-dried products irrespective of the technique employed in predicting Tc.
Now for those of you who maybe have been working in lyophilization for a number of years, you know, in the earlier days, the method for optimizing a cycle was just pretty much open the door to the freeze dryer, put your product in, and push a button, and hope that at the end of the day you get something that at least works, and again, you may burn through 20 to 30 runs, and a lot of product.
Now, these two trap water very differently, so identification is key. In the crystalline phase, you may have, you know, for a completely crystalline system, your product at the end of primary drying is already 99.99 percent dry.
Well, there are a couple of different ways we can do it. These first four bullet points represent what we call thermal analysis. Again, DSC is modulated, DSC is by far the gold standard, and we always couple that with freeze dry microscopy experiments.
Now, so I look at the DSC data, I look at the microscopy data, and come up with what we call a critical temperature, where we can freeze dry the product safely, or where we actually lose the product, and then we back that temperature down a little bit to make sure we are drying safely.
So, what have we learned from the thermal characterizations? It tells us a couple of things: Is this system crystalline, amorphous, or is it a mixture of both, of the two? It tells us the critical temperatures, or at least tells us glass transition of eutectic melting temperature.
Now, again, the system will also tell us if we have a metastable system, and if we need an annealing step. We can actually do annealing with the systems again, this is a little bit outside of the scope here.
One of the main things is that the glass transition can be anywhere between 5-15 difference, than our actual collapse temperature, and if we are able to freeze dry warmer, we exponentially increase the rate of drying of our product.
Again, thermal analysis (studies) tells 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.
Next, we will actually have a motorized valve on the system. This actually controls the pressure inside of the actual thermal stage itself. Via the software, we can actually regulate the pressure inside, just like you do on a standard pilot-scale, and/or production-scale freeze dryer.
Next, all of our systems are provided with a polarized light microscope. On a polarized light microscope, we have a polarizing condenser, analyzer, first order red compensator, objectives, and your camera.
On the condensers, there is a special lens that needs to be added on to it; this is a long working distance extension lens. This is needed because our sample is higher up inside of this stage, and to therefore properly align the microscope, this extension lens needs to be added on to the condenser, so that we can make sure that our microscope is aligned properly.
Like any other analytical piece of equipment, a microscope cannot be calibrated, however, we need to make sure that it is aligned properly so all the light possible forms through our sample so we can resolve this as much as we can.
Next we have our analyzer, and our analyzer is the same material as a polarizer, except we have to be able to distinguish one from the other, so the one the light passes through first is always known as our polarizer; the one that the light passes through after our sample is always known as our analyzer.
Again, we have our different objectives on here. The objectives must have a working distance greater than 4.5 mm. Systems that we provide are a Nikon polarized light system, which includes a 5X, 10X, 20X, and 50X objective.
The way that you would sample load is that you would first introduce the sample holder into the stage. We will then place a single drop of silicon oil onto the block, inside of the ring. Once we place our 16mm quartz window, which is our substrate, what happens then is that the oil fills in underneath that 16mm quartz window, and therefore, you have even thermal contact from the block onto the substrate. In case there are any imperfections on the quartz coverslip, or if there are any micro-scratches on the block, all of that gets filled in with the oil so that you have that even thermal contact.
The objective of this study was to characterize the thermal properties of systems containing various ratios of amorphous and crystalline components using both differential scanning calorimetry (DSC) and freeze-drying microscopy. The glycine/sucrose system was used as a model system, since it is routinely used in protein formulations. DSC analysis revealed that the addition of glycine to sucrose solutions resulted in a decrease in the glass transition (T'g) of the system. The T'g of a pure sucrose solution (7% w/v) decreased from -32.3 to -51.5 degrees C for a mixture containing a sucrose/glycine ratio of 2:5. The glass transition of the sucrose/glycine mixture decreased linearly as more glycine was added to the system. This decrease in glass transition resulted in severe collapse during freeze-drying of these mixtures above T'g. However, collapse was not observed during freeze-drying if the DSC thermogram of the sucrose/glycine mixture exhibited a transition resulting from recrystallization of the amorphous glycine. Mixtures having a sucrose/glycine ratio of 3:4 and 2:5 had a glass transition of -48 degrees C and -51.5 degrees C, respectively. Despite their low glass transition temperatures, these samples freeze-dried readily at a product temperature > T'g using a fast freeze-drying cycle (primary drying at a shelf temperature of +20 degrees C and chamber pressure of 100 mTorr) without any sign of collapse. The crystallization of the amorphous glycine from the frozen mixture of sucrose and glycine provided support during freeze-drying which prevented the macroscopic collapse of the final product. Freeze-drying microscopy visually revealed the crystallization and allowed for prediction of cake quality upon lyophilization. Although the freeze-drying microscope is not as sensitive as the DSC in detecting all transitions (it cannot detect a glass transition), it clarifies the interpretation of DSC, and together they provide valuable information regarding the relevance of each of the transitions to the final freeze-dried product elegance.
Freeze-drying (lyophilization) removes most of the water in a sample under low temperature and vacuum conditions, providing a dry, active, shelf-stable, and readily soluble product. It is therefore a widely used technique in the manufacture of protein biomolecules for therapeutic and diagnostic applications.2 As well as stabilizing drug products for transport and preventing degradation, freeze-drying reduces the volume of the sample, thus it requires less storage space and can be stored under ambient conditions.
Lyophilized drugs are representing an increasing proportion of the market, with the upward trend particularly strong in the biologics segment, where innovation and drug development is advancing rapidly. In addition, the complexity and strict storage conditions needed to preserve the biological drug activity make freeze-drying a popular formulation technique.
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