Sterilization in Parenteral Dosage forms (Moist Heat Sterilization)

 

What is acceptable appearance in Lyophilized products-II

Dried Product Between Vial and Stopper

During the manufacturing of the lyophilized products, while filling bulk solutions into vials, splashing may occur. Sometimes while manual loading of the tray with filled vials may leave a few droplets of bulk solution around the neck of the vial and even on the stopper. After completion of Lyophilization, If we observe, in these vials at the bottom cake may be intact and elegant. But at the top, but there are chances of freeze-dried product at the neck of the vial or between vial and stopper.

Dried product between vial and stopper

Product Sticking to stopper and neck of vial

Impact: This is a critical defect as the container closure integrity failure may occur resulting in comprise of sterility of the drug product. Also, due to the product adhering to the stopper, there are chances of low deliverable doses. Hence the vials shall be rejected. 

Mitigation: Avoid the solution of Splashing over the neck walls of the vial. Use Siliconized vials / Rubber stoppers if possible. Using larger wide neck vials and stoppers.

Cracked cake and cake shrinkage

Cake shrinkage refers to the apparent loss of cake volume typically by the cake pulling away from the walls and perhaps the bottom of the vial. Often, inverting the vial causes the cake to come loose from contact with the vial. 

Cake shrinkage

Cracked Cake

Shrinkage may be the first manifestation of collapse. However, most cake shrinkage is not associated with collapse. 

Mitigation: For the development scientist, it is important to ensure that cake shrinkage is not associated with collapse. This can be done easily by trial cycles at a range of conditions to be sure that collapse is not contributing to cake shrinkage. Thermal analysis of the frozen formulation and the dried cake (e.g., differential scanning calorimetry, freeze-drying microscopy), online process analytical technology methods (e.g., manometric temperature measurement, thermocouples), or offline characterization methods (e.g., X-ray powder diffraction, residual moisture, SSA) can help to distinguish between unintended collapse or unavoidable shrinkage.

Impact: Both cake shrinkage and cracked cake should not be considered defects, because the underlying physics has to do only with the mechanical properties of the cake, which are dependent on the formulation and primary container. Neither visual attribute has ever been shown to have any impact on CQAs of freeze-dried products.

Reference and more information:

http://dx.doi.org/10.1016/j.xphs.2017.03.014

What is acceptable appearance in Lyophilized products-I

Hello folks, 

Working with lyophilized products is always interesting. If lyo cycle went well and everything goes fine then no issues. But, Lyophilization is more tricky when unexpected variations happened during lyo cycle. This results in different kinds of issues majorly, related to appearance of product. 

Normally, after lyophilization the resulting product may be an intact cake or powder or some kind of flakes depending on the type and concentration of ingredients in the formulation. During visual inspection, there will be collection of acceptable appearance vials along with  separation of vials with defects. So, it is important to determine which cake is acceptable and which is not. Sajal patel et.al have published a wonderful article about what is an acceptable cake after lyophilization and what is not acceptable. 

Let us have a look at few cakes with issues.

After lyophilization, drug products are subjected to visual inspection. These visual inspection parameters include cake appearance, extraneous particulate matter, and assessment of minor, major, and critical defects of the lyophilized drug product. The appearance of an ideal lyophilized cake should be uniform and elegant without any defects and should have adequate mechanical strength to avoid cake disruption during handling and distribution. 

Collapsed cake:

This is the most common defect found in lyophilized product. This may or may not be a critical defect. Also, the collapse is crumbling of lyophilized cake. This may be partial or complete. 

Left vial: Complete collapse, Centre vial: Partial collapse, Right vial: No Collapse.

Reason : Most of the time, Collapse may occur during primary drying when product temperature exceeds the collapse temperature (Tc) of formulation. 

Impact: Increased residual solvent content (If organic solvents are part of formulation), increased reconstitution time. Some studies indicating, it is just cosmetic issue and there is nothing to do with stability of drug product. 

What to do: The impact of collapse on critical quality attributes shall be evaluated and the decision shall be taken as per the impact on CQA. 

Sometimes, the collapse may not necessarily at the initial stages of drug product but may also occur at the storage during stability particularly at high temperatures. This is attributed due to the glass transition temperature  is near to the product storage temperature.

various degrees of cake collapse during storage under stressed conditions.

 

How to avoid Collapse: Just keep the product temperature below collapse temperature or glass temperature of the product during primary drying. 

Melt back: 

Not to mean it as a synonym of collapse. Many opinions on melt back as either melting of frozen matrix during the freeze-drying process or collapse. But actually melt back is the presence of ice at the starting of secondary drying. it means the removal of ice during primary drying is not complete. During the secondary drying, the remaining ice is not removed. When the process is finished, the ice will melts and wet the product. Sometimes melt back could also be a form of collapsed cake.

Melt back from Right to left on the order of proportion

Ice crystals after primary drying

Reason & impact: Meltback usually indicates poor formulation and process understanding and, hence, product with meltback is rejected.

Rectification: The process should be modified by setting longer primary drying time to ensure complete removal of ice from the cakes without increasing the primary drying temperature.

Product Blow out : 

It is some part of partially dried or completely dried product coming out of the vial during either primary drying or secondary drying. Sometimes the product may stick to the stopper also. It is also called as product ejection. 

Blow out of product during primary drying

Blown out product 

Ejected product deposited on stopper region 

Reason: It is more common in formulations containing organic solvents such as t-butanol or ethanol, as well as in formulations containing a very low level of total dissolved solids, resulting in a poorly cohesive cake (i.e., a more fragile cake).

Impact: Product ejection is generally indicated by solid material in the “shoulder” and neck area of the vial. It should be considered as a critical defect because the solid material is likely also present between the sealing surfaces of the vial and the closure, which would compromise the sterility assurance of the product (container closure integrity failure) and the deliverable dose, thereby endangering the patient.

Rectification: By optimizing the primary drying duration and decreasing the vacuum of the primary drying step. Increasing the concentration of dissolved solids can be considered.  Using higher capacity vial. By combination of two or more before said practices the blow out can be avoided. 

End point determination in primary drying in Lyophilization

Hi Folks,

Primary drying is longest step of lyophilization process. So, it has to be optimized to make the shortest possible lyophilization recipe for the product.

Till previous posts, we came upto setting the conditions for primary drying step of Lyophilization process.
Once the parameters were set, how long the step of primary drying shall be continued???

The answer is till the frozen ice is removed completely (Some authors describe till removal of unbound moisture) from the formulation.

ok, fine. How we will come to know that there is no ice left in the formulation???

The question need an exact answer. It requires evaluation of quantity of ice in lyophilization chamber.
Now the question is How it can be measured?

For all the above questions, there is multiple answers together called as 'Determination of End point of primary drying".

Here it is.....

The primary drying time is directly related to the ice sublimation rate and is determined by numerous factors, including chamber pressure, shelf temperature, heat transfer coefficient of vials, fill volume, and product resistance.

As per the literature, The end point of primary drying can be detected by several different methods. At the end of primary drying, there is no ice present in vials (i.e., no ice sublimation and no heat
removal by sublimation); thus, the product temperature increases to the shelf temperature, and the vapor composition in the freeze-drying chamber changes from essentially all water vapor during primary drying to mostly air or nitrogen. Thus, product temperature data indicate the end point of
primary drying when the product temperature approaches the shelf temperature. Normally, the product temperature as a function of time shows a steep increase in temperature at the end of primary drying, followed by a plateau.

Techniques based on gas composition in the product chamber:
1. Comparative pressure measurement (i.e., Pirani vs.capacitance manometer)
2. Dew point monitor (electronic moisture sensor)
3. Process H2O concentration from tunable diode laser absorption spectroscopy (TDLAS)
4. Lyotrack (gas plasma spectroscopy)

Others:

5. Product thermocouple response
6. Condenser pressure
7. Pressure rise test (manometric temperature measurement (MTM) or variations of this method)


Comparative pressure measurement (i.e., Pirani vs.capacitance manometer): 
Comparative pressure
measurement also works well. The thermal conductivity pressure gauge (Pirani pressure gauge) is calibrated against air and shows higher vapor pressure during primary drying because the thermal conductivity of water vapor is about 1.5 times that of air or nitrogen. At the end of primary drying, the pressure difference between the thermal conductivity pressure gauge (Pirani gauge) and capacitance pressure gauge (MKS Baratron gauge, which measures actual pressure) decreases and approaches zero. The point where the Pirani pressure starts to sharply decrease (i.e., onset) indicates that the gas composition is changing from mostly water vapor to nitrogen; i.e., sublimation is “essentially” complete.

Dew point monitor (electronic moisture sensor):
 Dew point sensors, which can detect the vapor composition change or the relative humidity in the freeze-drying chamber, shows a sharp dew point decrease at the end of primary drying due to the vapor compositions in the chamber changing from almost 100% water vapor to essentially 100% nitrogen.

Process H2O concentration from tunable diode laser absorption spectroscopy (TDLAS): 
Tunable diode laser absorption spectroscopy (TDLAS) directly measures the water vapor concentration (molecules/ cm3) in the duct connecting the chamber and the condenser. The TDLAS unit is commonly installed with two laser beams, one directed with and the other directed against the vapor flow. TDLAS works on basic spectroscopic principles measuring absorption of radiation by water vapor to monitor the trace concentration of water vapor during primary drying that are used to determine the end point of primary drying. The point where water concentration starts decreasing sharply (i.e., onset) indicates that the gas composition is changing, and hence sublimation is

“essentially” complete.

 Lyotrack (gas plasma spectroscopy): 
This method is the latest addition to the online monitoring devices for freeze-drying and is manufactured by Alcatel Vacuum Technology, France. Lyotrack is based on optical emission spectroscopy and measures water vapor concentration during the drying process.

Product thermocouple response:
The end point of primary drying can also be determined from the product thermocouple response, assuming the vials containing the thermocouples are representative of the batch as a whole. Product temperature approaching the shelf temperature set point  is commonly taken as an indication of the end of primary drying.

Condenser pressure: 
During primary drying, most of the gas in the chamber is water vapor, and because the total
vapor flux is high, a high ΔP (difference between chamber and condenser pressure) develops to remove the water from the chamber. However, once primary drying is over, ΔP. decreases (i.e., condenser pressure (Pcond) increases since chamber pressure (Pc) is held constant). The condenser
pressure reflects mostly the partial pressure of nitrogen in the condenser. The purpose of the nitrogen bleed is to control the chamber pressure at the desired set point. The point where condenser pressure starts increasing (i.e., onset) indicates that the sublimation is “essentially” over since the high mass transfer portion of the process (i.e., sublimation) is largely over (Fig. 2). A capacitance manometer installed in the condenser reads the condenser pressure.

Pressure rise test (manometric temperature measurement (MTM) or variations of this method):
MTM is a procedure to measure the product temperature during primary drying by quickly isolating the chamber from the condenser for a short time (≈25 s) and analyzing the pressure rise during this period. This analysis yields vapor pressure of ice at the sublimation interface, the product temperature, and the mass transfer resistance of the dried product.

However, the data obtained measure the vapor pressure of ice accurately only as long as the system remains in primary drying. At the end of primary drying, there is little or no pressure rise because all ice is gone, and hence the calculated “vapor pressure of ice” becomes equal to the chamber pressure. Thus, a close approach of the calculated vapor pressure of ice to the chamber pressure forms the basis of the criterion for end of primary drying. The vapor pressure of ice determined by a fit of pressure rise data to the MTM equations approaches the chamber pressure when no ice remains (Refer: X. C. Tang, S. L. Nail, and M. J. Pikal. Freeze drying process optimization by manometric temperature measurement, 2001 AAPS Annual Meeting, Denver, Colorado, 2001).


Thats all about the methods for determination of end point of primary drying.


With regards,
Teja Ponduri

Pressure in Primary drying phase of Lyophilization

Hi Folks,

Now Let's have look on Primary drying phase aka sublimation phase that will follow when the frozen material, placed under vacuum, is progressively heated to deliver enough energy for the ice to sublime.

During this very critical period a correct balance has to be adjusted between heat input (heat transfer) and water sublimation (mass transfer) so that drying can proceed without inducing adverse reactions in the frozen material such as back melting, puffing, or collapse.

A continuous and precise adjustment of the operating pressure is then compulsory to link

the heat input to the “evaporative possibilities” of the frozen material.

Primary drying is carried out at low pressure to improve the rate of ice sublimation. The chamber pressure impacts both heat and mass transfer and is an important parameter for freeze-drying process design. Chamber pressure should be well below the ice vapor pressure at the target product temperature to allow a high sublimation rate.

The sublimation rate is proportional to pressure difference between the vapor pressure of ice and the partial pressure of water in the chamber (Pi), this difference being the driving force for ice sublimation. Pi is essentially the same as chamber pressure during primary drying. At given product

temperature (i.e., given ice vapor pressure), the smallest chamber pressure gives the highest ice sublimation rate.

So, as per the above concept let us set the vacuum set point for primary drying step. (temperature selection done as per previous posts).

A recommended approach is to first set the system pressure using the vapor pressure of ice table. 

Using the vapor pressure of ice table is a scientific way to determine an appropriate pressure for freeze drying. A general guideline is to choose a system pressure that is 20% to 30% of the vapor pressure of ice at the target product temperature. When the vacuum level set point is deeper than the vapor pressure of ice at the current product temperature, sublimation can take place. 

Herewith attached the vapor pressure of Ice table.

During the Initial days of the Lyophilization utilization, it is believed that higher the vacuum the better the sublimation process could be. 

However, very low chamber pressure may cause problems, such as contamination of product with volatile stopper components or pump oil, and also produce larger heterogeneity in heat transfer, thereby giving larger product temperature heterogeneity between vials. In most applications of practical interest, the chamber pressure varies from 50 to 200 mTorr. It is difficult to maintain consistently chamber pressure much below 50 mTorrr, and there is little reason to use pressures much higher than 200 mTorr. It has been reported that moderate chamber pressure (100–150 mTorr) gives optimal homogeneity of heat transfer in a set of vials.Therefore, the optimum chamber pressure is a compromise between high sublimation rate and homogenous heat transfer.

Thats how vacuum is set for primary drying folks.

Yours,
Teja Ponduri

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