Freeze drying

[COLOR="rgb(139, 0, 0)"]Freeze drying
Drying is an important operation in primary pharmaceutical manufacture(ie.the synthesis of actives ) as it is usually the last stage of manufacturing before packaging and it is important that the residual moisture is rendered low enough to prevent drug deterioration during storage and ensure free flowing properties during use.
Its equally important (and probably encountered more frequently ) in secondary (dosage form) manufacture following the commonly performed the operation of wet granulation during the preparation of granules prior to tablet compaction .

Drying is defined as the removal of all or most of the liquid by supplying latent heat to cause thermal vaporization, ie. A liquid is converted to a vapor . in the majority of cases the liquid will be water but volatile solvents such as isopropanol may also need to be removed in a drying process. The physical principles are similar regardless of nature of the liquid although volatile solvents are normally recovered by condensation rather than being vented into atmosphere .The toxicity and flammability of organic solvents pose safety consideration

Freeze drying
Freeze, drying is a process used to dry extremely heat-sensitive materials. It allows the drying, without excessive damage, of proteins, blood products and even microorganisms, which retain a small but significant viability.
In this process the initial liquid solution or suspension is frozen, the pressure above the frozen state is reduced and the water removed by sublimation. Thus a liquid-to-vapour transition takes place, as with all the previous driers discussed, but here there are three states of matter involved: liquid to sold, then solid to vapour. The theory and practice of freeze drying is based, therefore, on an understanding and application of the phase diagram for the water system.

The phase diagram for the water system is shown in figure 26.14. The diagram consists of three separate areas, each representing a single phase of water, either solid, liquid or vapour. Two phase can coexist along a line under the conditions of temperature and pressure defined by any point on the line. The point 0 is the one unique point where all three phases can coexist, and is known as the triple point. Its coordinates are a pressure of 610 Pa and a temperature of 0.0075°C.

The lines on the phase diagram represent the interphase equilibrium lines, which show:
1. The boiling point of water as it is lowered by reduction of the external pressure above the water ( BO in Fig. 26.14) ;
2. The variation of the melting point of ice on reduction of the external pressure above it. There is a very slight rise in the melting point (AO);
3. The reduction of the vapour pressure exerted by ice as the temperature is reduced (CO).

Application of the phase diagram of water to freeze drying
the freeze drying of products such as blood plasma, although simple in theory, presents a number of practical problems.
1.The depression of the freezing point caused by the presence of dissolved solutes means that the solution must be cooled to well below the normal freezing temperature for pure water and it is usual to work in the range -10 to -30 C . in part this is becauseit is obviously not pure water that is being dried, and thus the presence of dissolved solutes will shift the pure-water phase diagram
2. sublimation can only occur at the frozen surface and is a slow process
(approximately 1 mm thickness of ice per hour).for all but very small volumes the surface area must therefore be increased and the liquid thickness prior to freezing be reduced in order to reduce the thickness of ice to be sublimated.
3. At low pressures large volumes of water vapour are produced which must be rapidly removed to prevent the pressure rising above the triple point pressure
4.The dry material often needs to be sterile, and it must be prevented from regaining moisture prior to final packing.

Uses of Freeze Drying:
The method is used for products that cannot be dried by any other heat method. These include biological products, for example some antibiotics, blood products, vaccines (such as BCG, yellow fever, smallpox), enzyme preparations (such as hyaluronidase) and microbiological cultures. The latter enables specific microbiological species and strains to be stored for long periods with a viability of about 10% on reconstitution.

Cryo- and lyo-protection of proteins by stabilizers

both freezing and dehydration can induce protein denaturation. To protect a protein from freezing (cryoprotection) and:or dehydration (lyoprotection) denaturation, a protein stabilizer(s) may be used. These stabilizers are also referenced as chemical additives , co-solutes , co-solvents , or excipients. variety of protein stabilizers are presented for cryo- and lyo-protection .

*stabilizers for cryo- and lyo-protection:
Nature protects life from freezing or osmotic shock by accumulating selected compounds to high concentrations ( >1 M) within organisms. These accumulated compounds are known as cryoprotectants and osmolytes, which are preferentially excluded from surfaces of proteins and act as structure stabilizers .

*Sugars : polyols :
Many sugars or polyols are frequently used nonspecific protein stabilizers in solution and during freeze-thawing and freeze-drying. They have been used both as effective cryoprotectants and remarkable lyoprotectants. In fact, their function as lyoprotectants for proteins has long been adopted by nature ( anydrobiotic organisms contain high content of disaccharides ) . The level of stabilization afforded by sugars or polyols generally depends on their concentrations. A concentration of 0.3 M has been suggested to be the minimum to achieve significant stabilization. Since freezing is part of the freeze-drying process, high concentrations of sugars or polyols are often necessary for lyoprotection. They demonstrated that saccharides protects the protein by direct interaction with the protein and a concentration of saccharides sufficient to form a monomolecular layer on the protein surface was the minimum to achieve the maximum stabilization.

On the other hand, increasing sugar:polyol concentration to a certain level may eventually reach a limit of stabilization or even destabilize a protein during freeze-drying, which was apparently attributable to sticky, pliable, and collapsed formulation structure . depending on the formulation composition, concentration and physical properties of the stabilizer, and its compatibility with the protein. The ineffectiveness of larger saccharides suggests that protein stabilization by sugars may depend on their glucoside chain lengths, and a long chain length may interfere with intermolecular hydrogen-bonding between stabilizing sugars and proteins. In many cases, disaccharides appear to be the most effective and universal stabilizers among sugars and polyols . In comparison
to sucrose, trehalose seems to be a preferable lyoprotectant for biomolecules, because it has a higher glass transition temperature . The higher glass transition temperature of trehalose arises at least partly from the formation of trehalose–protein–water microcrystals, preventing water plasticizing the amorphous

Other properties of trehalose are also considered to be advantageous, which include (1) less hygroscopicity, (2) an absence of internal hydrogen bonds, which allows more flexible formation of hydrogen bonds with proteins, and (3) very low chemical reactivity. In reality, the relative stabilization effect of these two sugars seems to be depend on both the protein and sugar concentration.

. Polymers:
Polymers have been used to stabilize proteins in solution and during freeze-thawing and freeze drying One of the favorable polymers used in the history of protein drug development was serum albumin. It has been used as both a cryoprotectant and lyoprotectant. Human serum albumin (HSA), due to its effective inhibition of protein surface adsorption and general stabilization of proteins during lyophilization, was used in formulating freeze-dried hydrophobic cytokines.
Many protein products on the market, such as Betaseron®, Epogen®, Kogenate®, and Recombinate™ contain albumin . However, the ever-increasing
concern about the potential contamination of serum albumin with blood-borne pathogens limits its future application in protein products. Therefore,
rHA has been recommended recently to replace serum albumin as a protein stabilizer. Nevertheless, the ultimate solution is to develop albumin-free formulations for protein pharmaceuticals. In addition to albumin, other polymers also have been used . The level of protein stabilization afforded by these polymers depends on structure and concentration of both the polymer and the
protein. dextran, PVA hydroxypropyl methylcellulose (HPMC) or gelatin

Stabilization of proteins by polymers can generallybe attributed to one or more of these polymer properties: preferential exclusion, surface activity, steric hindrance of protein–protein interactions, and:or increased solution viscosity limiting protein structural movement. In recent years, additional properties of polymers have been implicated in stabilizing proteins during freeze-thawing
and freeze-drying. Polymers such as dextran have been reported to stabilize proteins by raising the glass transition temperature of a protein formulation
significantly and by inhibiting crystallization of small stabilizing excipients such as sucrose. The inhibition of crystallization of small molecules is apparently due to polymer-induced viscosity increase .
On the other hand, polymers may cause phase separation during freezing, adversely affecting protein stability . Certain polymers
may destabilize proteins during lyophilization
due to steric hindrance, preventing efficient hydrogen bonding with proteins.

*Protein itself :
Protein aggregation in solution is generally concentration dependent . It has been suggested that increasing protein concentration to higher than 0.02 mg ml_1 may facilitate potential protein aggregation . Increasing protein concentration increases aggregation of many proteins in solution. In contrast, proteins at higher concentrations are often more resistant against both freezing and lyophilization-induced protein denaturation / aggregation. The activity recovery of many labile proteins after freeze-thawing correlates directly with initial protein concentration
Freeze thawing. The mechanisms of proteins’ self-stabilization during freezing and:or lyophilization have not been clearly delineated. Proteins are polymers, and therefore, at least some of the above-discussed stabilization mechanisms for polymers may be applicable to proteins’ self-stabilization. Recently, two hypotheses have been reiterated to explain the concentration-dependent protein stabilization upon freezing :
First, unfolding of proteins at high concentrations uring freezing may be temporarily inhibited by steric repulsion of neighboring protein molecules.
Second, the surface area of ice-water interface formed upon freezing is finite, which limits the amount of protein to be accumulated and denatured at the interface. In addition, favorable
protein–protein interactions (possible formation of dimers or multimers) may contribute to the increased protein stability at high concentrations, as observed for thermophilic proteins .
The formation of ice-water interfaces during freezing may cause surface denaturation of proteins. Surfactants may drop surface tension of protein solutions and reduce the driving force of protein adsorption and:or aggregation at these interfaces. Low concentrations of nonionic surfactants are often sufficient
to serve this purpose due to their relatively low critical micelle concentrations. Other stabilization mechanisms were also
proposed, such as assistance in protein refolding
during thawing and protein binding, which may
inhibit protein–protein interactions. Tween 80 is one of the commonly used surfactants for protein stabilization during freezing. . Other nonionic and ionic surfactants have also been reported in cryoprotection of proteins.
2.1. Lyophilization process
Lyophilization (freeze-drying) is the most commonprocess for making solid protein pharmaceuticalsThis process consists of two major steps: freezing of a protein solution, and drying of the frozen solidunder vacuum. The drying step is further dividedinto two phases: primary and secondary drying.The primary drying removes the frozen water andthe secondary drying removes the non-frozen‘bound’ water . Lyophilization generates a variety of stresses,which tend to destabilize or unfold:denature anunprotected protein. Different proteins toleratefreezing and:or drying stresses to various degrees.Freeze-thawing of ovalbumin at neutral pH didnot cause denaturation .Some proteins can keep their activity both duringfreezing and drying processes.However, many proteins cannot stand freezingand:or drying stresses.

Denaturation stresses during lyophilization
The lyophilization process generates a variety
of stresses to denature proteins. These include (1)
low temperature stress; (2) freezing stresses, includingformation of dendritic ice crystals, increasedionic strength, changed pH, and phase
separation; and (3) drying stress (removing of the protein hydration shell).


  • dr.Hazemdr.Hazem مدير عام
    تم تعديل 2010/05/02
    A. Low temperature stress :

    The nature of cold denaturation has not beensatisfactorily delineated. Since solubility of nonpolargroups in water increases with decreasingtemperature due to increased hydration of thenon-polar groups, solvophobic interaction inproteins weakens with decreasing temperature.The decreasing solvophobic interaction in proteins canreach a point where protein stability reaches zero,causing cold denaturation.
    B. Concentration effect:
    Freezing a protein solution rapidly increases theconcentration of all solutes due to ice formation.Thus, all physical properties related to concentrationmay change, such as ionic strength and relativecomposition of solutes due to selectivecrystallization. These changes may potentially
    destabilize a protein.Generally, lowering the temperature reduces therate of chemical reactions. However, chemicalreactions may actually accelerate in a partiallyfrozen aqueous solution due to increased soluteConcentration,The increase in the rate of a chemical reactionin a partially frozen state could reach several orders of magnitude relative to that in solution The reported oxygen concentration in a partially frozen solution at _3°C is as high as 1150times that in solution at 0°C. The increased oxygen concentration can readily oxidize sulphydryl groups in proteins. If a proteinsolution contains any contaminant proteases, concentration upon freezing may drastically accelerate protease-catalyzed protein degradation.

    C. Formation of ice-water interface:
    Freezing a protein solution generates an ice-water=interface. Proteins can be adsorbed to the interface, loosening the native fold of proteins and resulting in surface-induced denaturation. Rapid (quench) cooling generates a large ice-water interface while a smaller interface is induced by slow cooling

    D. pH changes during freezing :
    Many proteins are stable only in a narrow pH Range. At extreme pHs, increased electrostatic repulsion between like charges in proteinstends to cause protein unfolding or denaturation. Thus, the rate of protein aggregation is strongly affected by pH, Moreover, the solution pH can significantly affect the rate of many chemical degradations in proteins*Freezing a buffered protein solution may selectively crystallize one buffering species, causing pH changes, This can lead to a significant pH drop during freezing, which then denatures pH-sensitive protein. The pH drop during freezing can potentiallyaffect storage stability of lyophilized proteins.
    E. Phase separation during freezing:
    Freezing polymer solutions may cause phase separation due to polymers’ altered solubilities at lowtemperatures. Freezing-induced phase separation can easily occur in a solution containing two incompatible polymers. Several strategies have been proposed to mitigate or prevent phase separation-induced proteindenaturation during freezing. These include use ofalternative salts, adjustment of the relative composition of polymers to avoid or to rapidly pass over a temperature regionwhere the system may result in liquid–liquidphase separation and chemical modification of the protein such as pegylation.
    F. Dehydration stresses :
    Proteins in an aqueous solution are fully hydrated. A fully hydrated protein has a monolayer water covering the protein surface, which is termed the hydration shell. Generally, the water contentof a lyophilized protein product is less than 10%. Therefore, lyophilization removes part of the hydration shell. Removal of the hydration shell may disrupt the native state of a protein andcause denaturation. A hydrated protein, when exposed to a water-poor environment during dehydration, tends to transfer protons to ionized carboxyl groups and thus abolishes as manycharges as possible in the protein. The decreased charge density may facilitate protein–protein hydrophobic interaction,causing protein aggregation.
    Water molecules can also be an integral part of an active site(s) in proteins. Removal of these functional water molecules during dehydration easily inactivates proteins. Lastly, dehydration during lyophilization may cause significant difference in moisture distribution in different locations of a product cake. The uneven moisture distribution may lead to possible localized over drying, which may exacerbate dehydration- induced protein denaturation.

    Design of a robust lyophilization cycle — a
    step-by-step analys
    The purpose of designing a robust lyophilizationcycle for protein pharmaceuticals is to obtaina consistent, stable, and esthetically acceptable product. To achieve this goal, a number of parameters that directly determine or characterize a lyophilization cycle need to be determined ordefined. These parameters should include glasstransition temperature (Tg% ):collapse temperature (Tcol), cooling rate, drying rate, and residual moisture content.

    Stages of the freeze dying process:
    Freezing stage

    The liquid material is frozen before the application of vacuum to avoid frothing, and several methods are used to produce a large frozen surface.
    Shell freezing . this is employed for fairly large volumes such as blood products. The bottles are rotated slowly and almost horizontally in a refrigerated bath. The liquid freezes in a thin shell around the inner circumference of the bottle. Freezing is slow and large ice crystals form, which is a drawback of this method as they may damage blood cells and reduce the viability of microbial cultures.In vertical spin freezing, the bottles are spun individually in a vertical position so that centrifugal force forms a circumferential layer of solution, which is cooled by a blast of cold air. The solution super cools and freezes rapidly, with the formation of small ice crystals.

    Centrifugal evaporative freezing. This is a similar method, where the solution is spun in small containers within a centrifuge. This prevents foaming when a vacuum is applied. The vacuum causes boiling at room temperature and this removes so much latent heat that the solution cools quickly and snap freezes. About 20% of the water is removed prior to freeze drying and there is no need for refrigeration. Ampoules are usually frozen in this way, a number being spun in a horizontal angled position in a special centrifuge head so that the liquid is throuwn outwards and freezes as a wedge.

    Vacuum applications stage
    The containers and the frozen material must be connected to a vacuum source sufficient to drop the pressure below the triple point and remove the large volumes of low-pressure vapour formed during drying. Again an excess vacuum is normal in practice, to ensure that the product in question is below its triple point.
    Commonly a number of bottles or vials are attached to individual outlets of a manifold, which is connected to a vacuum.

    Sublimation stage
    Heat of sublimation must be supplied. Under these conditions the ice slowly sublimes, leaving a porous solid which still contains about 0.5% moisture after primary drying.
    Primary drying. Primary drying can reduce the moisture content of a freeze dried solid to around 0.5%. further reduction can be affected by secondary drying. During the primary drying, the latent heat of sublimation must be provided and the vapor removed.
    Heat transfer. Heat transfer is critical: insufficient heat input prolongs the process, which is already slow, and excess heat will cause melting.Pre-frozen bottles-of blood, for example – are placed individually heated cylinders, or are connected to a manifold when heat can be taken from the atmosphere.
    Shelf-frozen materials are heated from the drier shelf, whereas ampoules may be left on the centrifuge head or may be placed on a manifold, but in either case heat from the atmosphere is insufficient. In all cases the heat transfer must be controlled, as only about 5W m-2 K-1 is needed and overheating will lead to melting. It is important to appreciate here that although a significant amount of heat is required there should be no significant increase in temperature – the added heat should be sufficient to provide the latent heat of sublimation only and little sensible heat.

    Vapour removal. The vapor formed must be continually removed to avoid a pressure rise that would stop sublimation. To reduce sufficiently it is necessary to use efficient vacuum pumps. usually two stage rotary pumps on the small scale, and ejector pumps on the large scale. On the small scale, vapour is absorbed by a desiccant such as phosphorus pentoxide, or is cooled in a small condenser with solid carbon dioxide, mechanically refrigerated condensers are used on the large scale. For vapour flow to occur the vapour pressure at the condenser must be less than that at the frozen surface, and a low condenser temperature is necessary. On the large scale vapour is commonly removed by pumping, but the pumps must be of a large capacity and not affected by moisture. The extent of the necessary pumping capacity will be realized from the fact that, under the pressure conditions used during primary drying, 1g of ice will form 1000L of water vapour. Ejector pumps are most satisfactory for this purpose

    Rate of drying. The rate of drying in freeze drying is very slow. The ice being removed at a rate of about only 1 mm depth per hour. The drying rate curve illustrated in figure 26.15 shows a similar shape to a normal drying curve, the drying being at constant rate during most of the time.
    Computer control enables the drying cycle to be monitored. There is an optimum vapour pressure for a maximum sublimation rate and the heat input and other variables are adjusted to maintain this value.
    Continuous freeze drying is possible in modern equipment, where the vacuum chamber is fitted with a belt conveyor and vacuum locks, but despite these advances the overall drying rate is still slow.

    Secondary Drying:
    The removal of residual moisture at the end of primary drying is performed by raising the temperature of the solid to as high as 50 or 60 C. A high temperature is permissible for many materials because the small amount of moisture remaining is not sufficient to cause spoilage.
    Attention must be paid to packaging freeze-dried products to ensure protection from moisture.
    Containers should be closed without contacting the atmosphere. If possible, and ampoules, for example, are sealed on the manifold while still under vacuum.
    Otherwise, the closing must be carried out under controlled atmospheric conditions.

    As a result of the character of the process, freeze drying has certain special advantages:
    1. Drying takes place at very low temperatures, so that enzyme action is inhibited and chemical decomposition, particularly hydrolysis, is minimized.
    2. The solution is frozen such that the final dry product is a network of solid occupying the same volume as the original solution. Thus, the product is light and porous.
    3. The porous form of the product gives ready solubility.
    4. There is no concentration of the solution prior to drying Hence, salts do not concentrate and denature proteins, as occurs with other drying methods.
    5. As the process takes place under high vacuum there is little contact with air, and oxidation is minimized

    There are two main disadvantages of freeze drying:
    1. The porosity, ready solubility and complete dryness yield a very hygroscopic product. Unless products are dried in their final container and sealed in situ, packaging requires special conditions.
    2. The Process is very slow and uses complicated plant, which is very expensive. It is not a general method of drying, therefore, but is limited to certain types of valuable products which, because of their heat sensitivity, cannot be dried by any other means.