Annealing in freeze drying

Hi everyone,

In this session, let us understand the concept of Annealing in freeze drying.

• What is annealing
• What happens during annealing
• When Annealing is required
• The concept of annealing (If required, at what conditions the annealing to be done)
• More about annealing

What is annealing:

'Annealing' is defined as process of transient increase in product temperature from initial set point to higher or lower set point, and then bringing the product temperature back to original set point.

It is simply holding the product at a temperature above the final freezing temperature for a defined period to crystallize the potentially crystalline components (usually, crystalline bulking agent) in the formulation during the freezing stage.

In annealing we warm and hold a sample above it’s glass transition temperature (but below the eutectic and/or ice melting temperature) and allowing the glass to relax and crystallize completely. 

Mannitol and glycine are routine examples. 

What happens during annealing:

The annealing temperature should be between the T_g of amorphous phase melting temperature to give a high crystallization rate and complete crystallization.

Annealing involves a process of warming a frozen material below the total ice melting point to bring about phase changes 

– Crystallization of excipient: must be below the eutectic melting point of crystallized material

– Ostwald Ripening of ice: must be above T_g’ or T_eu of solution to have free water present

Ostwald Ripening of ice: A thermodynamic ‘adsorption’ phenomena (Involves Thermo= heat, dynamic=movement) occurs in a mixture where smaller molecules shrink and disappear. Where as larger molecules grows bigger and bigger (Of course, at the expense of smaller crystals).

As the system tries to lower its overall energy, molecules on the surface of a small (energetically unfavorable) particle will tend to detach and diffuse through solution and then attach to the surface of larger particle. Therefore, the number of smaller particles continues to shrink, while larger particles continue to grow. 

Concentration of the molecules around the interface of smaller particle is larger than the average concentration in bulk solution, resulting in net flux of molecules flowing from particle to the solution

phase, leading to shrinking of the small particle. Reversely for the larger particle, where the local concentration around the interface is lower than average concentration in bulk solution, resulting in net flux of molecules flowing from the solution phase to the particle, thereby leading to growth of the large particle.

'Ostwald ripening: Larger particles grow at the expenses of smaller particles'

For more details please follow the link https://www.youtube.com/watch?v=dh4q55aaXWw

The frozen sample temperature shall be increased till annealing temperature and hold for enough time for completion of crystallization. The annealing time depends on depends on the mass ratio and properties of the bulking agent used.

A high mass ratio of bulking agent to other solutes (>80% of total solute, recommended) crystallizes much faster than a lower ratio (<50% of total solute, not recommended) (Tang and Pikal, unpublished).

A low annealing temperature may tend to produce high crystallinity because, supersaturation is higher at low temperature, but the crystallization rate may be too low because of high viscosity.

The optimum annealing conditions are a compromise between crystallinity and crystallization rate.

For Mannitol or Glycine, a temperature of −20 or −25°C and an annealing time of 2 h or longer are suggested if the fill depth is 1 cm or more.

When Annealing is required:

An annealing step is frequently necessary to allow efficient crystallization of the crystalline bulking agent, such as mannitol or glycine present in the formulation at high concentration.

Failure to crystallize the bulking agent has the potential of depressing the T_g and compromising storage stability by crystallizing from the solid during storage.

If the bulking agent crystallizes during primary drying, vial breakage may result, which is common if a high fill depth of concentrated mannitol is used.

Vial breakage can be prevented by crystallization of mannitol during freezing using slow freezing or by avoiding a temperature lower than about −25°C until the mannitol has completely crystallized.

Completion of crystallization may be facilitated by annealing.

The concept of Annealing:

For a successful Freeze drying of any material, the pre- requisites are, 

• Formation of Ice
• Crystallization of solutes and/or Formation of glass 
• 
  
  

 

A typical Freezing protocol involving the Annealing or Thermal treatment will consist of the following steps: 

• First freezing the product at low temperature
• Warming it gradually to a predetermined temperature well above the glass transition temperature
• Holding there for a enough period of time to allow any metastable state to crystalize out
• And then cooling it again to suitable temperature before initiating primary drying

The concept can be more understandable by an example. [‘A Study of the Phase Transitions in Frozen Antibiotic Solutions by Differential Scanning Calorimetry’ by Larry Gatlin and Patrick P. Deluca]

• The sample drug chosen for the study was ‘cefazolin sodium’- a cephalosporin antibiotic.
• Initially, low temperature (to simulate freezing conditions) DSC thermograms of the drug were generated.From the DSC thermograms obtained, they found some observations.
• At first, an endothermic shift occurring at -20°C which is glass transition (represented as Point A).
• Further, An irreversible exothermic shift was beginning at -11°C, which may be an indicative of recrystallization of Ice and solutes (represented as Point B).
• Later, melting of Ice (Endothermic shift) occurred at -4°C (represented as Point E)

From the available data, the similar composition was rewarmed to -6°C (Which is just 2°C below the melting point of formulation) after cooling resulted in a formation of a crystalline product. Here the annealing temperature is -6°C.

Glass transition temperature	Melting point of formulation	Annealing temperature
-20°c	-4°c	-6°c

Observation: Annealing temperature is +16 °C  than Glass transition temperature and -2°C than Melting point of formulation. So, the Annealing temperature should be chosen in such a way that, the Formulation may show transition of solid phase to enhance the flow or movement of molecules and should not result in melting of frozen formulation. 

For the above example, the typical freezing protocol was provided below,

Temperature	Ramp (minutes)	Hold (minutes)	Vacuum
-40°C	45	90	-
-6°C	45	120	-
-40°C	45	120	-

More about annealing:

Annealing often has effects beyond crystallization of solutes.

Annealing above the glass transition temperature of T_g causes growth of ice crystals, which decreases the product resistance to flow of water vapor and results in shorter primary drying time.

Also, the product specific surface area is reduced, which decreases the water desorption rate in secondary drying and may lead to increased residual moisture content in the final product or demand longer secondary drying.

Annealing conditions can be studied using either frozen solution X-ray diffraction or DSC procedures to evaluate the development of crystallinity.

This is about annealing introduction folks, lets meet again..Till then bye bye...


Yours,
Teja Ponduri