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

Concept of Freezing in lyophilization-III (Freezing behavior of solutes in aqueous solutions during freeze drying)

Hello folks,

Till now we have gone through the various aspects of freezing with respective to lyophilization in pharmaceutical industry.

Wee know that,

• Freezing is the first step of a freeze drying process, and the characteristics of the frozen matrix strongly affect drying rates at primary and secondary stages.
• At the end of the freezing step, about 65% to 90% of the initial moisture is in the frozen state and the rest remains at the adsorbed state in many cases.
• The freezing temperature, freezing rate and supercooling degree are all important factors influencing the overall drying time and product quality.
• Based on the physical and chemical properties of material, the freezing protocol can be optimized to produce the most favorable freeze drying results in terms of both high product quality and short drying time.

As majority of pharmaceutical products are Aqueous based, now let’s have a look at Freezing of Aqueous formulations.

Freezing of Aqueous solutions:

The liquid material being frozen displays one of the three behavior (from freezing session ii) i.e., Crystalline/ amorphous/ mixture of both. 

• The liquid phase suddenly solidifies (eutectic formation) at a temperature depending on the nature of solids in solutions for crystalline substances.
• In Amorphous substances, the liquid phase does not solidify (glass formation), but rather it becomes more and more viscous until it finally takes the form of a very stiff substance, and becomes a highly viscous liquid.
• In the mixture of crystalline and amorphous substances, there may be partial eutectic formation and partial glass formation depending on the proportion of the kind of substances. 
• Before proceeding for the freeze concentration, lets have a brief about behavior of water with respective to various temperatures and pressure combinations in the perspective of freeze drying. (Usually described under titles ‘Phase diagram of water’ or ‘Triple point of water’).

The physical state of water molecules (H₂O) depends on the temperature and pressure surrounding them. We all know that, At Ambient temperature and pressure water exist as liquid. By keeping the pressure at one atmosphere, and

Ø  Increasing the temperature, water exists as a gas (Moisture)

Ø  Decreasing the temperature, water exists as a Solid (Ice)

Freeze drying takes place below the triple point of water (Temperature of 0.0098°C and water vapour pressure of 4.58 mm Hg, not to confuse as the lyophilizer chamber pressure). 

Lets have a look at the Phase diagram for an idealized, simple lyophilization cycle. 

·A liquid sample first is cooled the solution’s freezing point.

· At this point, it is thermodynamically favorable to form a new solid phase composed of pure ice. Once ice begins to form, the remaining components of the solution in the unfrozen phase become increasingly more concentrated, as shown in Figure.

· The combination of increased concentration and lower temperatures causes the viscosity of the non-ice phase to increase until, at a glass transition point termed T_g', [Glass transition temperature T_g' corresponds to a change in the viscosity of solution from a viscous liquid to a glass or an essentially solid solution of solute in water] the solution becomes so viscous that further freezing of water is kinetically blocked.

· Further temperature decreases below T_g' have no additional concentrating effects.

· Primary drying occurs under vacuum at a temperature below the glass transition temperature.

· After primary drying, the temperature is increased to effect secondary drying.

·  Final storage temperature after secondary drying is below the glass transition line.

Freeze concentration

• If a solution is cooled below the normal freezing point without freezing, the solution is said supercooled. 
• For aqueous solution, the degree of supercooling temperature [The degree of supercooling refers to the difference between the equilibrium freezing point and the temperature at which ice crystals first form in the sample] can be in a range of 10 to 15 °C below 0 °C, depending on the nucleation temperature of ice. 
• Following the time scale of freezing, a sudden increase in temperature, T_f, indicates the crystallization of ice due to the release of latent heat as shown in  Figs. 4 and 5 . 

• For the first case, crystalline components, which have the least solubility in the formulation, form a mixture with crystalline water, and the temperature increases to the eutectic crystalline temperature, T_e. 
• A eutectic is defined as an intimate physical mixture of two or more crystalline solids, having then the same physical properties as if it were one component. 
• However, a multi-component mixture often presents no T_e , because in this freezing stage, the molecules diffusion is reduced dramatically, which, on the other hand, is essential for crystallization. 
• Therefore, one of the most important parameters to optimize the freeze drying process is the reversible transition between viscous and glassy state, termed the glass transition temperature of the freeze-concentrated solution, T_g’. 

Franks illustrated the freezing behaviour of a   sucrose-water system as shown in Fig. 6. The solute phase is concentrated from an initial solid content of 5% to about 80%, which suggests that most of separation in the freeze drying process occurs in the freezing stage, but still a large fraction of unfrozen water exists. The sucrose-water system does not precipitate as a crystal phase when the solution is cooled down to the eutectic point, but remains in a thermodynamically unstable solution. Below T_g’, the system behaves like a solid.

When the material is further cooled, more liquid water is converted into ice and all interstitial fluid in the vicinity concentrates ultimately until it crystallizes or the viscosity of the system is so high that the system transforms into a solid amorphous state. 

A more practical discussion of freezing, annealing, and lyophilization is aided by viewing the process through the “supplemented phase diagram” described by MacKenzie is provided below.

Eutectic formation of crystalline substances: 

• A typical binary eutectic system is the sodium chloride-water system as illustrated in phase diagram of Nacl-water system. Understanding the behaviour of this system is useful for a conceptual understanding of material science in freeze drying.

• In the phase diagram, Line ‘ab’ indicates, the product temperature decreases to below the equilibrium freezing temperature of the product nothing but freezing point depression curve of water in the presence of sodium chloride.
• In the phase diagram, Line ‘ab’ indicates, the product temperature decreases to below the equilibrium freezing temperature of the product nothing but freezing point depression curve of water in the presence of sodium chloride.
• At the point ‘b’ nucleation of ice crystals occurs. As nucleation and crystal growth of ice begins at ‘b’, energy is released (The latent heat due to crystallization of ice) and the temperature increases to T_f  (Freezing temperature of solution).
• Further, cooling continues with ice crystal growing and the interstitial fluid becoming more concentrated.
• At the point ‘c’, crystallization of concentrated interstitial fluid is initiated: An eutectic mixture of crystalline Nacl/ice. when eutectic crystallization is initiated, the temperature of the product increases to the eutectic temperature T_e.
• After eutectic crystallization is completed at the point T_e, no more liquid is present and no changes in microstructure of frozen system take place. Then, the product temperature decreases more rapidly toward the shelf temperature.
• The line ‘bc’ represents the solubility of sodium chloride in water. The intersection of the two lines at point b is the eutectic melting temperature, which for sodium chloride/ice is 21.5°C.
• Freezing of a 5% solution of sodium chloride in water is described by line ‘defgh’. At room temperature of point ‘d’, the system is entirely liquid.
• As the solution cools, ice appears at point e in the absence of supercooling. As the system cools, ice continues to crystallize and the solution becomes more concentrated with sodium chloride.
• At point ‘f’, two phases are present, ice and a freeze concentrated solution of sodium chloride in water.
• This freeze concentrated solution has the composition given by point ‘i’, which is in equilibrium with ice.
• At point ‘g’, the solution is saturated with respect to sodium chloride, and solid sodium chloride begins to precipitate.
• It is only below the eutectic temperature that the system is completely solidified (point ‘h’).
• Similarly, if the freezing path is across line ‘bc’ with an initial concentration of the solution being between ‘b’ and ‘c’, the solid sodium chloride precipitates first rather than ice. 
• Other example of binary eutectic systems is glycine-water, which has similar freezing histories.Here are the substances that form a eutectic mixtures in aqueous solutions. 

ØGlycine

ØMannitol

ØSodium carbonate

ØDibasic sodium phosphate

ØCitric acid

Glass transition of Amorphous substances

• In the most cases, the solute does not readily crystallize during freezing.
• The first part of curve is the same as in the case of eutectic compounds.
• Then crystallization does not occur, but a slight change in slope of the temperature vs time curve is observed when a material forms an amorphous phase, it remains as a liquid below the normal freezing point but eventually goes through a rapid increase in viscosity as temperature falls.
• This transition is defined as the glass transition since the material is glassy. 
• A glass is a true solid that has the chemical composition of the crystalline solid but does not have the ordered molecular structure of the crystalline solid.For amorphous systems, glass transition temperature (T_g) corresponds to a change in the viscosity of solution from a viscous liquid to a glassy semi solid or solid.
• T_g is important for amorphous solute as T_e for crystalline solute. It represents the maximum allowable product temperature during the primary drying.
• If product temperature exceeds the glass transition temperature, the product will undergo collapse.

• For the crystalline compounds, the interstitial material consists of a mixture of eutectic ice and crystalline solute.
• when the ice is removed by sublimation, a crystalline solid with very little water is left.
• For the amorphous systems, the interstitial glassy material must be rigid enough to support its own weight after the ice is removed in order to keep the microstructure established during freezing.
• Lets see the behavior of sucrose solution.

• The line ab represents the freezing temperature of water as a function of solute concentration.
• Instead of the solute crystallizing at point b the interstitial material remains as liquid or freeze concentrate and continues along line ‘b’ is T_g.
• Ice crystals continue to grow, and the freeze concentrate become more concentrated and more viscous.
• The family of curves shown by the dashed lines are iso-viscosity curves, i.e., combinations of solute concentration and temperature that result in the same fluid viscosity.
• The solid line is the glass transition point of amorphous solid as a function of water content.
• As freezing proceeds, the freeze concentrate becomes more viscous until the system reaches point T_g and the growth of ice crystal stops.
• At the point T_g (the glass transition temperature of the freeze concentrate) the interstitial fluid changes from a viscous liquid or rubber to an elastic solid.
• The concentration of unfrozen water in glass is represented by W_g.

Examples of Amorphous substances:

• Dextran
• Fructose
• Gelatin
• Sorbitol
• Maltose
• Trehalose
• Lactose
• Glucose

The glass transition temperature of a material is strongly dependent on the moisture content.
Generally, the highest moisture content at the beginning of drying results in the lowest T_g, while the lowest moisture content at the end of drying leads to the highest T_g. Thus, there is not one single value of T_g but rather a range of T_g for a frozen material corresponding to a range of residual moisture content. At a particular moisture content, the glass transition temperature of a material is termed T_g’.

Hope the session is useful to have a glance on freezing behavior of crystalline and amorphous substances during freezing drying.

Here with signing off...

yours, Teja Ponduri...

Concept of Freezing in lyophilization-II

Hello folks,

In the last session i.e., concept of freezing in lyophilization -I, we have came to know about the importance of freezing, phenomena of nucleation, reasons for inability to freeze/freeze dry.

Lets further understand what actually happens in freezing of bulk solutions when loaded into lyophilizer.

The freezing step during which the material is hardened by low temperatures. In the freezing phase of the process the product is often cooled to -40 °C or lower and held for a period of time until the system is completely solid, with all “freezable water” converted to ice.

For understanding purpose, let us consider bulk solution is an aqueous solution (Water containing solids dissolved in it). When the bulk solution temperature is cooled from ambient temperatures to sub zero temperatures, water freezes in the first step, the dissolved components in the formulation remain in the residual liquid, a phase termed the freeze-concentrate. At the point of maximal ice formation, the freeze concentrate solidifies between the ice crystals that make up the lattice. Under appropriate lyophilization conditions, the ice is removed by sublimation during primary drying, leaving the remaining freeze-concentrate in the same physical and chemical structure as when the ice was present. 

During this very critical period all fluids present become solid bodies, which may be,

• Crystalline solids
• Amorphous solids
• Mixture of both amorphous and crystalline solids (Partially amorphous /partially crystalline)

Most often, water gives rise to a complex ice network, but it might also be imbedded in glassy structures or remain more or less firmly bound within the interstitial structures. Solutes do concentrate and might finally crystallize out. At the same time, the volumetric expansion of the system might induce powerful mechanical stresses that combine with the osmotic shock given by the increasing concentration of interstitial fluids.

At the most fundamental level, to achieve a lyophile with acceptable macroscopic appearance, the primary objective for crystalline materials is not to exceed their eutectic temperature and that for amorphous materials is not to exceed their glass transition temperature or collapse temperature between the initial cooling phase and the end of the sublimation process.

For the crystalline solutes, the interstitial material consists of a mixture of eutectic ice and crystalline solute. when ice is removed by sublimation, a crystalline solid with very little water is left.

For amorphous system, the interstitial glassy material must be rigid enough to support its own weight after the ice is removed in order to keep the micro-structure established during freezing.

However, often a formulation will contain a mixture of crystallizing and amorphous components, in which case, the critical temperature may not be so obvious. One approach is to assume complete phase separation will occur, assess the individual components separately, then aim to cool the starting material to below the lowest of the critical temperatures and maintain it below this temperature until the end of the sublimation process.

Effect of freezing rate on lyophilization:

Do we think there is something on freeze drying due to freezing rate?? The answer is YES.

Generally, with slower rate of freezing, larger ice crystals form. With rapid freezing rate, smaller crystals.

Slow freezing can subject the bulk solutions for longer periods of time, permitting maximum crystal growth that leads to larger sized crystals.

An aqueous solution cooled at rapid cooing and slow cooing rates, the resulting ice crystals were examined microscopically and the results were provided below.

Which is better, rapid freezing or slower freezing?

The crystal size formed during freezing can significantly affect the dissolution rate of the dried material.

A fast ice growth also helps to prevent the denaturation of proteins (If present) which may result from prolonged exposure to strong concentrations of salts because of slow ice growth.

Rapid cooling results in small ice crystals, useful in preserving structures to be examined microscopically, but resulting in a product that is, more difficult to freeze dry.

Slower cooling results in large ice crystals and less restrictive channel in the matrix during the drying process.

The main pores in the solid residue after freeze drying are those left by the sublimation of pure ice and they form the principal channels for the escape of vapor.

During sublimation, very small ice crystals form smaller pores and pathways, which are more restrictive to vapour flow than those formed after slower freezing.

The appropriate cooling cycles must be determined in order to obtain an appropriate structure of the frozen mass, which is a function of the rate of freezing and the final freezing temperature.

Now you decide, whether you freeze your product at rapid rate or slower rate…….

In the next session will meet you again…

Teja ponduri signing off.....Bye, Bye…