Determining conditions for Primary drying of Lyophilization- Part III (FDM)

Hi Pharma folks,

A Sample done with DSC, the result tells us, what is the thermal event , is it a glass transition, is it eutectic melt, and what are the critical temperatures that are associated with it. Glass transition  doesn’t mean that’s going to collapse the sample.Sometimes we may have a glass transition that occurs, and we see no collapse. So this is the beauty of using both the microscope and the DSC, in that the microscope complements and supports the data that we get from the DSC.

In Freeze dry microscopy tells us  where that sample physically loses structure.

What actually happens in Freeze dry microscopy?

The sample upon exposure to heat during DSC, may undergoes various thermal events. But, we cannot view/visualize  those events. There comes Freeze dry microscopy (FDM). With FDM, we can view the various thermal events of sample directly with change in temperature as well as pressure combination. Nothing but, we can simulate the conditions of Freeze drying at micro level and see what happens to the sample.

In FDM, very tiny sample quantity (typically Less than 2 µL) will be placed on thermally controllable stage and completely enclosed in a chamber in order to hold the vacuum applied. (just imagine a lyophilizer chamber with shelves). The sample will be undergone cooling and followed by applying vacuum and further increasing the temperature at a slow phase to see at what temperature the collapse of the sample is visible.

FDM is nothing but, a direct examination of stages of freeze drying using a special microscope and a thermal stage (Thermally controllable stage as mentioned in above paragraph).

What a Freeze dry microscope contains?

Sample Preparation:

The bulk solution which has to be lyophilized should be the sample for Freeze dry microscopy. The sample which has to be lyophilized should be utilized in FDM. Below diagram depicts the sample preparation at a glance.

Initially, the sample stage will be like this.

Now, let us know about parts of a stage.

The sample stage is provided with a vacuum ports, and for stage adjustment purpose will have X and Y manipulators visible on exterior view and also a sample holder will be present inside which will be place onto the stage with the help of sample door lock.

Coming to the interior of stage, a 22 mm silver block containing 1.3 m  light aperture onto which the sample will be placed, and is connected with below parts as provided in the picture.

• Liquid nitrogen inlet
• Liquid nitrogen outlet
• Thermocouple leads (for temperature determination)

Before sample loading, below procedure shall be followed.

• Ensure silver block is cleaned and have enough silicone oil on the aperture. 
• Place a 16 mm cover slip and a sample separator and then 2 µL bulk solution followed by 13 mm cover slip.

Once sample is prepared, the stage is closed with a lid and is ready for the evaluation.

Operation:

• Initially the Freeze dry microscope should be calibrated with known concentration of calibrating substance such as NaCl. 
• Once, the collapse temperature was attained within a specified range, then actual sample shall be evaluated.
• The sample will be ran through the steps of freezing similar to lyophilization recipe but at a faster rates during freezing. 
• Once sample was frozen completely, then focus has to be adjusted such that sample was clearly visible clearly. 
• Then vacuum pump was switched on and slightly temperature was increased to start primary drying at a rate that all the thermal events are clearly recorded. 
• If any rough idea about the range of the transition temperature then at that temperature, drying at a slow rate shall be performed.
• The images will be captured such that at material changes at all temperature points with each 0.1°C for accurate recording of collapse.    
• Once collapse is identified, it can be taken a screenshot and video/images can be recorded.
• Then the sample shall be brought back to room temperature and   vacuum pump to be swithced off, and vacuum to be made to atmospheric pressure. 
• Stage shall be cleaned and closed with the lid. 
• The example FDM data is provided below which helps to interpret the data.

  

Note: 

Why colors ?

When we use a Ist order red compensator, depending on the material whether Isotropic or an-isotropic in presence of polarized light, the images obtained are colored as per the orientation of ice crystals frozen direction (IF ice crystal formed parallel to the direction of light path will show one color and if in perpendicular to light path then other color).

For complete concept please refer (Practical Application of FD Microscopy in Product Thermal Characterization, by Ruben Nieblas).

Thats all for the FDM, will post on primary drying concept in the next post.

Till then, take care.

Yours, 

Teja Ponduri

Determining conditions for Primary drying of Lyophilization- Part II (DSC-II)

Hi folks,
In the last blog "Determining conditions for Primary drying of Lyophilization- Part I" we have seen the utilization of Differential scanning calorimetry (DSC) to determine the glass transition temperature of formulation, that helps us to set shelf temperature during primary drying of lyophilization of pharmaceuticals.

Now lets see other useful information that can be obtained from DSC.

If the sample run through DSC, it makes sense that every possible information shall be obtained. Hence, lets have a glance on those information.

Crystallization: 
A Sample after glass transition, substances will have a lot of mobility and never stay in one position for very long time. But when they reached a specific temperature, it will give off enough energy to move into very ordered arrangements, which are called crystalline substances and they release heat. So it doesn't have to put out much heat to keep the temperature of the sample pan rising. This drop in the heat flow as a big peak in the plot of heat flow vs. temperature.

The temperature at the highest point in the peak is usually considered to be the crystallization temperature, or Tc. Also, the area of the peak can be measured, which tells us the latent energy of crystallization of the substance. But most importantly, this peak tells us that the substance can in fact crystallize. If 100% amorphous polymer is analysed, like polystyrene, this peak cannot be obtained, because such materials don't crystallize also, because the polymer gives off heat when it crystallizes, called as crystallization is an exothermic transition.

Also, liquid formulation during freezing step once crystallization occurs that may result in little sharp peak from base line of a DSC as shown in above figure.

Now lets see, what happens when the substance gets heated beyond its crystallization temperature. The resulting thermal transition is called Melting.

Melting:

When substance's melting temperature is reached, polymer crystals begin to fall apart, that is they melt. It comes out of their ordered arrangements, and begin to move around freely that can be spotted  on a DSC
plot The heat which polymer give off when crystallized is absorbed when reached at Tm. That is a latent heat of melting like latent heat of crystallization. When the polymer crystals melt, they must absorb heat in order to do so. Melting is a first order transition. This means that at the melting temperature, the polymer's temperature won't rise until all the crystals have melted. The heater under the sample pan has to put a lot of heat into the polymer in order to both melt the crystals and keep the temperature rising at the same rate as that of the reference pan.  This extra heat flow during melting shows up as a big dip on DSC plot, like this:

In a DSC curve, all the three phase transitions seen above Glass transition, Crystallization, melting are denoted in a single curve as shown below.

 Before concluding the topic of DSC, for a better understanding on variations in DSC curves due to  crystallization and melting process are provided below.

Conclusion:  
Even though DSC is useful for Tg determination, it should not be the one and only evaluation to determine the primary drying temperature. As any method has its own uses and limitatiions, DSC has below limitations.

• can not really control the rate of experiment (can only be checked but cant controlled)
• Dependent on too many parameters (Thorough understanding of analysis is required for analyst)
• Very sensitive to any changes 
• Result depends a lot from the operator (Well trained operator required)

That's all for today folks. Hope the content was useful.

Take care......

Regards,
Teja Ponduri