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...