Lyophilization Freeze Drying

During lyophilization the water of deeply frozen samples is removed by sublimation in vacuum. The structure of a probe is well conserved, especially if additives as sucrose or trehalose are added. In a sealed botde, the sample can be stored at RT for years. If the buffer constituents are to be removed also, the sample has to be dissolved in a volatile buffer (see Table 7.6). Reconstitute the sample by addition of water or, in the case of volatile buffers, by addition of a buffer of your choice. 

For optimal conditions it is necessary to freeze the sample very quickly in a methanol-dry ice bath or in liquid nitrogen and to store the frozen sample below -30 °C until the vacuum of less than 100 Pa (0.0145 psi) is reached and the lyophilization is finished. 

During the lyophilizing process, the sample has to get external heat because sublimation of ice withdraws heat resulting in cooling the probe, but avoid melting of the sample. 

Concentration of a sample in a SpeedVac, a centrifuge with open rotor running in vacuum, is not lyophilization because the sample is not frozen therefore, this method does not fit for macromolecules with defined structures. The reconstitution (dissolving) of protein samples concentrated in a SpeedVac is often more difficult and comparable to drying with air. But it is the method of choice for concentration of solutions of low-molar mass substances, e.g., peptides, since the sample is collected at the bottom of the tube by means of centrifugal force. 

Altman was the first to report microscopic investigations of freeze-dried tissue in 1890. Arrhenius, and later Becquerel, proposed that life originated by freeze-dried organisms that arrived on this planet from outer space and then were reconstituted by water. Becquerel calculated that if a dehydrated organism could live for one year at 10 C, then it could live for 70 billion years at -270 °C, the temperature of outer space. 

A freeze-dried material undergoes fewer adverse changes than those preserved by pickling, salting, canning, or normal heat dehydration. The product requires simpler storage and transportation systems, and if sealed under vacuum or an inert gas, usually retains most of its biological and physical characteristics indefinitely. 

The material to be freeze dried is first frozen to a temperature just below its lowest eutectic. A eutectic is the lowest temperature at which a mixture of two or more components will melt. This eutectic temperature can be determined in an unknown system by measuring the electrical resistance between two probes in the sample or by 

Bx = Brix, a measure of sugar concentration based on density. A hydrometer is used. One degree Brix equals the % by weight of sucrose in a water-sugar solution. 

A vacuum of from 4-6 torr (0.53 - 0.80 kPa) is common, and the heat loss by the subliming water usually will keep the material frozen. During small-scale laboratory operations the sample holders are left in the open air, but in large-scale commercial applications, heat must be applied to provide the sublimation energy at the rate Just below that required to keep the material frozen. To sublime 1 g of ice at 0 C requires 666 calories (2.78 kJ). This heat is provided by warm-water trays placed under the sample trays, by radiation from heated walls surrounding the sample trays, or by microwave warming. Sublimation from commercial warm water-heated trays occurs at the rate of 0.1-1.0 kg HjO/hr/m. Few systems are cooled below -30 °F, because the vapor pressure is then too low for rapid sublimation. 

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In pharmaceutical systems, both heat and mass transfer are involved whenever a phase change occurs. Lyophilization (freeze-drying) depends on the solid-vapor phase transition of water induced by the addition of thermal energy to a frozen sample in a controlled manner. Lyophilization is described in detail in Chapter 16. Similarly, the adsorption of water vapor by pharmaceutical solids liberates the heat of condensation, as discussed in Chapter 17. 

Before the extraction procedure may commence, the sample must be prepared in such a way that it is in a condition for extraction of the analyte(s). For analyzing sulfonamide residues in liquid samples such as milk, a pretreatment dilution step with water prior to direct fluorometric detection may be required (207). Dilution of milk with aqueous buffer (208) or sodium chloride solution (209) prior to sample cleanup has also been reported. For the analysis of honey a simple dissolution of the sample in water (210, 211) or aqueous buffer (212) is generally required. Semisolid samples such as muscle, kidney, and liver, require, however, more intensive sample pretreatment. The analyte(s) must be exposed to extracting solvents to ensure maximum extraction. The most popular approach for tissue break-up is through use of a mincing and/or homogenizing apparatus. Lyophilization (freeze-drying) of swine kidney has been carried out prior to supercritical-fluid extraction of trimethoprim residues (213). 

Concentration. This group consists of those processes in which water is removed and the dissolved substances are left behind. Examples are freeze concentration, lyophilization (freeze-drying), vacuum distillation, and membrane processes such as reverse osmosis and ultrafiltration. A common disadvantage of these methods is that inorganic species are concentrated along with the organic constituents. 

Ultrafiltration (qv) (uf) is increasingly used to remove water, salts, and other low molecular-weight impurities (21) water may be added to wash out impurities, ie, diafiltration. Ultrafiltration is rarely used to fractionate the proteins because the capacity and yield are too low when significant protein separation is achieved. Various vacuum evaporators are used to remove water to 20—40% dry matter. Spray drying is used if a powdery intermediate product is desired. Lyophilization (freeze-drying) is only used for heat-sensitive and highly priced enzymes. 

Reducing sugars, 4-8%,7 are known to be present in cane juice they are D-glucose and D-fructose. The acetylation of lyophilized (freeze-dried) normal cane juice solids followed by chromatography on a magnesium acid silicate of these acetates led to the isolation and proper identification of the sugars as crystalline derivatives.8... 

Bakaltcheva, I., Reid, T. Lyopilization of blood cells. International Conference on Lyophilization-Freeze-Drying, Amsterdam, October 2002, International Society of Lyophilization-Freeze-Drying,... 

Remove the organic solvent by lyophilization/freeze-drying or centrifugation under vacuum. 

The practical solution to the protein stability dilemma is to remove the water. Lyophilization (freeze-drying) is most commonly used to prepare dehydrated proteins, which, theoretically, should have the desired long-term stability at ambient temperatures. However, as will be described in this review, recent infrared spectroscopic studies have documented that the acute freezing and dehydration stresses of lyophilization can induce protein unfolding [8-11]. Unfolding not only can lead to irreversible protein denaturation, even if the sample is rehydrated immediately, but can also reduce storage stability in the dried solid [12,13]. 

Adjustments in pH lo maintain water solubility cun sometimes lead lo chemical stability problems. An example is indomelhacin (HA acid pK ,4.S). which is unstable in alkaline media. Therefore. Ihc preferred oral liquid dosage form is a suspension buffered at pH 4 to 5. Because this is near the drug s pK . only. W f will be in the water-soluble form. There is a medical indication requiring intravenous administration of indomelhacin to premature infants. The intravenous dosage form is the lyophilized (freeze-dried) sodium salt, which is reconstituted just prior lo use. 

The lipid was hydrated to a concentration of 20-mg/ml. The formulation was to be lyophilized (freeze-dried) by snap freezing in liquid nitrogen and sublimation of the water, resulting in dry and stable lipid vesicles. 

By early 2004, 15 million doses of smallpox vaccine, Dryvax, were available in the United States. Dryvax, a Wyeth product, is a live virus preparation of vaccinia. The vaccine comes as a lyophilized (freeze-dried) powder in 100 dose vials containing the antibiotics polymyxin B, streptomycin, tetracycline, and neomycin. Fifty percent glycerin, containing a small amount of phenol as a preservative, serves as the diluent for reconstitution (25). Studies have shown that diluting the vaccine in a 1 5 ratio could expand the supply without reducing vaccine efficacy (25). In addition, 200 million antibiotic-free doses are in production. The CDC Web site has detailed directions on how to reconstitute and administer the vaccine at http //www.bt.cdc.gov/agent/smallpox/vaccination/ administration.asp. 

Enzyme activity is preserved by cold storage. Lyophilized (freeze-dried) proteins are stored in a freezer or refrigerator, while dilute solutions are stored at 2-5 °C. Concentrated suspensions of enzymes in ammonium sulfate are usually stable for long periods at 2-5 °C. 

Lyophilization (freeze drying) is used in laboratory medicine for the preparation of calibrators, control materials, reagents, and to a lesser extent individual specimens for analysis. Lyophilization first entails freezing a material at -40 °C or less and then subjecting it to a high vacuum. Very low temperatures cause the ice to sublime solid nonsub-limable material, initially locked in an ice matrix, remains behind in a dried state. 

Lyophilization (freeze drying) may be used to prepare a dosage form that is to be reconstituted for injection. The solution to be concentrated is distributed into small vials, and specialized equipment is employed to freeze the samples and remove the solvent. For other purposes lyophilization is impractical on scale. 

Lyophilization (Freeze Drying) Lyophilization is most frequently used for heat-labile dosage forms that are unstable in aqueous formulation. The principle of lyophilization can be seen by reference to the phase equilibrium diagram for water (Fig. 15). Water at atmospheric pressure and ambient temperatures is stable in its liquid phase at lOO C the liquid phase attains an equilibrium with its vapor phase. Above 100 C water is stable in its vapor phase. At atmospheric pressures and 0 C the solid (ice) and liquid phases of water are in equilibrium with each other. At vacuum pressures a temperature (the eutectic point) can be reached where the three phases, solid, liquid, and vapor are all in equilibrium with each other. At even lower temperatures and pressures the solid phase comes into equilibrium with the liquid phase. The significance of this is that an aqueous solution can be concentrated by evaporation (sublimation) at low pressures without any necessity for significant heat input. 

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