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ISSN: 0975-766X CODEN: IJPTFI Available through Online Review Article www.ijptonline.com A REVIEW ON FREEZE DRYING PROCESS AND TECHNOLOGIES Rapolu Bharath kumar* 1 CMR College of Pharmacy, Kandlakoya, Medchal, Hyderabad, A.P, India. Email:bharath.rapolu@gmail.com Received on 01-09-2012 Accepted on 18-09-2012 Abstract Freeze drying, also known as lyophilization, is a process used to remove water or other solvents from various products thereby rendering the product relatively inactive and able to be stored for some period of time, typically without being refrigerated. Freeze-Drying has been essentially concerned with the preservation of unstable biochemicals, and, more particularly, of injectables. The very first issue has been, then, to secure their sterility and safeguard their potency during processing, storage and reconstitution. At the onstart most products were freeze-dried as such, either as a natural substance, such are blood plasma, biosynthetic isolates or antibiotics or else, for vaccines, as controlled concentrates of inactivated and/or living cells resulting from selected culture fermentation broths. Lyophilization requires the development of a unique recipe or cycle based on the thermal and physical characteristics of the product along with consideration of temperature and pressure relationships, phase changes and heat transfer. A basic understanding of the theory behind freeze drying is essential for the scientist to begin to design a working lyophilization process. Keywords: Freeze dryer, Lyophilisation, Sublimation. Introduction 1 Lyophilization is a process which extracts the water from foods and other products so that the foods or products remain stable and are easier to store at room temperature (ambient air temperature). Lyophilization is carried out using a simple principle of physics called sublimation. Sublimation is the transition of a substance from the solid to the vapour state, without first passing through an intermediate liquid phase. To extract water from products, the process of lyophilization consists of: 1. Freezing the food so that the water in the product become ice; IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2215

2. Under a vacuum, sublimating the ice directly into water vapour; 3. Drawing off the water vapour; 4. Once the ice is sublimated, the products are freeze-dried and can be removed from the machine. Biological materials often must be dried to stabilize them for storage or distribution. Drying always causes some loss of activity or other damage. Lyophilization, also called freeze-drying, is a method of drying that significantly reduces such damage. Because lyophilization is the most complex and expensive form of drying, its use is usually restricted to delicate, heat-sensitive materials of high value. Substances that are not damaged by freezing can usually be lyophilized so that refrigerated storage is unnecessary. (Important exceptions are mammalian cells, nearly all of which are destroyed by lyophilized.) Many microorganisms and proteins survive lyophilization well, and it is a favored method of drying vaccines, pharmaceuticals, blood fractions, and diagnostics. Some specialist food products are also lyophilized. They rehydrate easily and quickly because of the porous structure left after the ice has sublimed. (The word lyophilized is derived from the Greek "made solvent-loving") Occasionally materials are lyophilized to achieve a porous, friable structure rather than for preservation. Lyophilizers are sometimes used for concentration of delicate materials. The form of the product and the type of container it is to be freeze-dried in influence the type of lyophilizer needed and how it should be operated. Origin of Freeze Drying Freeze-drying was first actively developed during World War-II. Serum being sent to Europe for medical treatment of the wounded required refrigeration. Due to the lack of available refrigeration, many serum supplies were spoiling before reaching the intended recipients. The freeze-drying process was developed as a commercial technique that enabled serum to be rendered chemically stable and viable without having to be refrigerated. Shortly thereafter, the freeze dry process was applied to penicillin and bone, and lyophilization became recognized as an important technique for preservation of biologicals. Since that time, freeze-drying has been used as a preservation or processing technique for a wide variety of products. Some of the applications include the processing of pharmaceuticals, diagnostic kits, restoration of water damaged documents, river bottom sludge prepared for hydrocarbon analysis, ceramics used in the IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2216

semiconductor industry, viral or bacterial cultures, tissues prepared for analysis, the production of synthetic skins and restoration of historic/reclaimed boat hulls. Freeze Drying Process 1, 2 There are four stages in the complete drying process: pretreatment, freezing, primary drying, and secondary drying. Pretreatment Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent or increasing the surface area. In many instances the decision to pretreat a product is based on theoretical knowledge of freeze-drying and its requirements, or is demanded by cycle time or product quality considerations. Methods of pretreatment include: Freeze concentration, Solution phase concentration, Formulation to Preserve Product Appearance, Formulation to Stabilize Reactive Products, Formulation to Increase the Surface Area, and Decreasing High Vapor Pressure Solvents. Freezing In a lab, this is often done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature. This cycling process is called annealing. However, in the case of food, or objects with formerly-living cells, large ice crystals will break the cell walls (a problem discovered, and solved, by Clarence Birdseye), resulting in the destruction of more cells, which can result in increasingly poor texture and nutritive content. In this case, the freezing is done rapidly, in order to lower the material to below its eutectic point quickly, thus avoiding the formation of ice crystals. Usually, the freezing temperatures are between 50 C and 80 C. The freezing phase is the most critical in the whole freeze-drying process, because the product can be spoiled if badly done. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2217

Amorphous materials do not have a eutectic point, but they do have a critical point, below which the product must be maintained to prevent melt-back or collapse during primary and secondary drying. Primary drying During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the material for the water to sublimate. The amount of heat necessary can be calculated using the sublimating molecules latent heat of sublimation. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be slow (can be several days in the industry), because, if too much heat is added, the material s structure could be altered. In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates provide a surface(s) for the water vapour to re-solidify on. This condenser plays no role in keeping the material frozen; rather, it prevents water vapor from reaching the vacuum pump, which could degrade the pump's performance. Condenser temperatures are typically below 50 C ( 60 F). It is important to note that, in this range of pressure, the heat is brought mainly by conduction or radiation; the convection effect is considered to be inefficient. Secondary drying The secondary drying phase aims to remove unfrozen water molecules, since the ice was removed in the primary drying phase. This part of the freeze-drying process is governed by the material s adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0 C, to break any physicochemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal). However, there are products that benefit from increased pressure as well. After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed. At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2218

Figure.1. Laboratory freeze dryer Properties of freeze dried products 3 If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may be stored at room temperature without refrigeration, and be protected against spoilage for many years. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance. Freeze-drying protectants 3 Similar to cryoprotectants, some molecules protect freeze-dried material. Known as lyoprotectants, these molecules are typically polyhydroxy compounds such as sugars (mono-, di-, andpolysaccharides), polyalcohols, and their derivatives. Trehalose and sucrose are natural lyoprotectants. Trehalose is produced by a variety of plant, fungi, and invertebrate animals that remain in a state of suspended animation during periods of drought (also known as anhydrobiosis). Applications 3 Pharmaceutical and biotechnology Pharmaceutical companies often use freeze-drying to increase the shelf life of products, such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2219

stored, shipped, and later reconstituted to its original form for injection. Another example from the Pharmaceutical industry is the use of freeze drying to produce tablets or wafers. The advantage of which is less excipient and a rapidly absorbed and easily administered dosage form. Food industry Freeze-drying is used to preserve food and make it very lightweight. The process has been popularized in the forms of freeze-dried ice cream, an example of astronaut food. It is also popular and convenient for hikers because the reduced weight allows them to carry more food and reconstitute it with available water. Instant coffee is sometimes freeze-dried, despite the high costs of the freeze-driers used. The coffee is often dried by vaporization in a hot air flow, or by projection on hot metallic plates. Freeze-dried fruit is used in some breakfast cereal. Culinary herbs are also freeze-dried, although air-dried herbs are far more common and less expensive. However, the freeze-drying process is used more commonly in the pharmaceutical industry. Technological industry In chemical synthesis, products are often freeze-dried to make them more stable, or easier to dissolve in water for subsequent use. In bio-separations, freeze-drying can be used also as a late-stage purification procedure, because it can effectively remove solvents. Furthermore, it is capable of concentrating substances with low molecular weights that are too small to be removed by a filtration membrane. Freeze-drying is a relatively expensive process. The equipment is about three times as expensive as the equipment used for other separation processes, and the high energy demands lead to high energy costs. Furthermore, freeze-drying also has a long process time, because the addition of too much heat to the material can cause melting or structural deformations. Therefore, freezedrying is often reserved for materials that are heat-sensitive, such as proteins, enzymes, microorganisms, and blood plasma. The low operating temperature of the process leads to minimal damage of these heat-sensitive products. Processing 4, 5 The fundamental process steps are: IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2220

1. Freezing: The product is frozen. This provides a necessary condition for low temperature drying. 2. Vacuum: After freezing, the product is placed under vacuum. This enables the frozen solvent in the product to vaporize without passing through the liquid phase, a process known as sublimation. 3. Heat: Heat is applied to the frozen product to accelerate sublimation. 4. Condensation: Low-temperature condenser plates remove the vaporized solvent from the vacuum chamber by converting it back to a solid. This completes the seperation process. The first step in the lyophilization process is to freeze a product to solidify all of its water molecules. Once frozen, the product is placed in a vacuum and gradually heated without melting the product. This process, called sublimation, transforms the ice directly into water vapor, without first passing through the liquid state. The water vapor given off by the product in the sublimation phase condenses as ice on a collection trap, known as a condenser, within the lyophilizer's vacuum chamber. To be considered stable, a lyophilized product should contain 3% or less of its original moisture content and be properly sealed. Principles of Lyophilization 6 The material is first frozen and transferred to a drying chamber. During the drying stage, the material in the chamber is subjected to a high vacuum. Heat is applied carefully to the material, and a condenser is used in the chamber to collect the water. When water is leaving rapidly, its heat of vaporization is taken from the material and helps to keep it cool and safe. As the material dries, this cooling diminishes so that it is possible to overheat and damage the material. Heat supplies the energy necessary for sublimation of the water. An ice crystal is composed of pure water that is rather rigidly confined in a crystal lattice. The molecules have natural vibrations, however, so that extra thermal energy increases the probability of breaking free. When the water molecule breaks free, it diffuses through the already dried surface of the solid and sublimes. As the water molecules diffuse and sublime, the thickness of the dry outer surface of the specimen increases, and thus more energy is required to transport the molecules through the dry shell. The actual force driving water vapor from the drying boundary, through the dry shell and to the specimen surface, is a concentration gradient, and not, as some would assume, the vacuum sucking on the sample. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2221

Figure.2 As the molecules sublime and use up the latent thermal energy, the thermal reserves in the specimen are depleted and thus the probability of further sublimation decreases. Rate of transfer through the dried solids is low. The rate of drying of the specimen decreases until such time that so much external thermal energy would have to be supplied that the specimen may be harmed. Sublimation can continue safely if no heat is supplied, but drying time is greatly extended. The removal of the water vapor that reaches the specimen surface is critical to completion of the drying process. The water molecules that have successfully sublimed must be removed from the free space of the vacuum. The molecules move through the vacuum-induced free space and are trapped by condensation. Some condensers are plates, but a device known as a 'cold finger' is common. The 'cold finger' is a long thin condenser. Figure.3 IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2222

Wet samples can be frozen by placing them in a vacuum. The more energetic molecules escape, and the temperature of the sample falls by evaporative cooling. Eventually it freezes. About 15% of the water in the wet material is lost. The simplest form of lyophilizer would consist of a vacuum chamber into which wet sample material could be placed, together with a means of removing water vapor so as to freeze the sample by evaporative cooling and freezing and then maintain the water-vapor pressure below the triple-point pressure. The temperature of the sample would then continue to fall below the freezing point and sublimation would slow down until the rate of heat gain in the sample by conduction, convection, and radiation was equal to the rate of heat loss as the more energetic molecules sublimed away were removed. This simple approach creates numerous difficulties. When a material is frozen by evaporative cooling it froths as it boils. This frothing can be suppressed by low-speed centrifugation. Centrifugation also helps to dry faster by reducing material thickness and exposing a greater surface area. An alternative is to freeze the material before it is placed under vacuum. This is commonly done with small laboratory lyophilizers where material is frozen inside a flask. The flask is then attached to a manifold connected to the ice condenser. To speed the process the material can be shell-frozen by rotating the flask in a low-temperature bath, giving a large surface area and small thickness of material. For larger-scale equipment it is usual to place the material on product-support shelves inside the drying chamber, which can be cooled so that the material is frozen at atmospheric pressure before the vacuum is created. Without a controlled heat in[put to the sample its temperature would fall until drying was virtually at a standstill. For this reason it is usual to arrange a heat supply to the product-support shelves so that, after their initial use for freezing the product, they can be used to provide heat to replace the energy lost with the subliming water vapor and maintain the product at a constant low temperature. One milliliter of ice produces more than 1,000,000 ml. of water vapor at typical lyophilization cycle pressures. The more energy-efficient vacuum pumps cannot handle large quantities of water vapor. For this reason it is usual to fit a refrigerated trap (called the ice condenser) between the lyophilization chamber and the vacuum pump. Modern lyophilizers incorporate refinements. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2223

The most important are listed below: Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology Separated drying chamber and ice condenser to reduce cross-contamination Provision of an isolation valve between chamber and ice condenser to allow for end-point determination and simultaneous loading and defrosting Construction of the chamber and ice condenser as pressure valves to allow for steam sterilization at 121 C or higher Cooling and heating of the product -support shelves by a circulating intermediate heat-exchange fluid to give even and accurate temperature Additional instruments to control, monitor, and record process variables Movable product-support shelves to close the slotted bungs used in vials and to facilitate cleaning and loading Automatic control system with safety interlocks and alarms, duplicated vacuum pumps, refrigeration systems, and other moving parts to enable drying to proceed without endangering the product in the event of mechanical breakdown. Lyophilization Equipment: A lyophilizer consists of a vacuum chamber that contains product shelves capable of cooling and heating containers and their contents. A vacuum pump, a refrigeration unit, and associated controls are connected to the vacuum chamber. Chemicals are generally placed in containers such as glass vials that are placed on the shelves within the vacuum chamber. Cooling elements within the shelves freeze the product. Once the product is frozen, the vacuum pump evacuates the chamber and the product is heated. Heat is transferred by thermal conduction from the shelf, through the vial, and ultimately into the product. Lyophilization Container Requirements: The container in which a substance is lyophilized must permit thermal conductivity, be capable of being tightly sealed at the end of the lyophilization cycle, and minimize the amount of moisture to permeate its walls and seal. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2224

The enclosed reagents can only remain properly lyophilized if the container in which they are processed meets these requirements. Lyophilization Heat Transfer: Successful lyophilization is heavily dependent on good thermal conductivity. For this reason, containers used in the lyophilization process must be capable of meeting a number of heat-transfer requirements. Such containers should be made of a material that offers good thermal conductivity; should provide good thermal contact with the lyophilizer shelf, which is the source of heat during processing; and should have a minimum of insulation separating the source of heat from the product requiring heating. Poor thermal conductivity often results from the use of containers made of materials with low coefficients of heat transfer. It can also be caused by the shape, size, or quality of the container. It may come from thermal barriers, such as excessive amounts of material, which can act as insulation, preventing energy from being transferred to the point at which the frozen ice and dried product interface. Poor thermal conductivity often results in a product that is not successfully lyophilized. In a serum vial, the surface of the frozen cake must sublime first to allow the ice vapor to escape. Thereafter, the sublimation front moves as the drying proceeds. Generally, the sublimation front simultaneously moves downward toward the bottom of the serum vial and inward toward the center of the frozen cake (the core). If sublimation is not controlled and it cannot be controlled when significant thermal barriers exist then portions of the chemicals may actually be vacuum-dried rather than freeze-dried. The processed product will then not possess the defined and reproducible characteristics of a properly lyophilized material, such as maximized retention of activity, optimized shelf life, rapid reconstitution, and a consistent finished cake. The rate of drying depends directly on the rate of application of that amount of heat which is required to supply the latent heat of sublimation of the ice. Much of the recent research on the freeze-drying process has, therefore, IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2225

been directed into investigating methods of heat transfer and identifying the factors which limit the rate of heat transfer. In a freeze-drying system one had a water vapour pressure difference between the water vapour pressure of the drying material and the condenser, a resistance to the flow of vapour and a rate of flow. This may be expressed thus : Vapour pressure difference/resistance to flow = Rate of flow. As the vapour pressure of ice is a function of temperature, the formula may be rewritten : VP. T, (drying material) - VP. T, (condenser)/resistance to flow = Rate of flow = Constant K x watts. If you wish to dry at a low temperature, then VP (T 1 T 2 ) must be kept small so that the drying temperature approximates to the condenser temperature, and if drying is to be fast, the heat input must be high. This, in turn, means that we must keep all obstruction to the flow of vapour as low as possible. Comparison with Liquid-Phase Drying 6 Lyophilization gives the opportunity to avoid denaturation caused by heating the product, by maintaining it frozen throughout drying. This is the most obvious advantage over liquid-phase drying. Equally important is that in liquid-phase drying there is an undesirable shrinkage and concentration of active constituents that causes damage as well as a movement of these constituents to the surface of evaporation, where they form a dense, impermeable skin that inhibits drying, and later, rehydration. Such effects can be avoided by spray drying, but this requires brief exposure to temperatures around 100 C. Further advantages of lyophilization for parenteral products are that the wet material can be accurately dispensed and can be sterile filtered just before filling into final containers so that particulate and bacterial contamination is reduced. Thus, the principle advantages of lyophilization as a drying process are: Minimum damage and loss of activity in delicate heat-liable materials Speed and completeness of rehydration Possibility of accurate, clean dosing into final product containers IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2226

Porous, friable structure Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology The principle disadvantages of lyophilization are: High capital cost of equipment (about three times more than other methods) High energy costs (2-3 times more than other methods) Long process time (typically 24 hour drying cycle) Lyophilization should be used when the product meets one or more of the following criteria: unstable; heat liable; minimum particulates required; accurate dosing needed; quick; complete rehydration needed; high value. Lyophilization of Blood Plasma 7 Lyophilization, or freeze drying, can be used to prepare plasma that will remain stable over prolonged periods of time. This is useful, for example, for preparation of lyophilized FVIII-defi cient plasma, which is normally stable for at least two and up to fi ve years when stored at -20 C or lower, and which is stable enough to survive short periods (up to seven days) at temperatures of 20 C 25 C. Suitable plasma Venous blood mixed with 0.105 0.109M sodium citrate in the proportion 9 parts blood, 1 part anticoagulant. Centrifuge at 1700 g for 10 minutes, pool as appropriate, and store at -55 C pending viral test results. Confirm negative results for anti-hiv 1 and 2, anti-hcv, and Hep B SAg. Materials 2 ml clear neutral glass vials with internally siliconized 13 mm neck. 13 mm freeze-dry stopper, grey. 13 mm fully tear-off seals. Freeze-dryer unit. N-2-Hydroxyethylpiperazine-N -2-ethanesulphonic acid. Method Thaw plasma rapidly at 37 C. Mix well. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2227

Add 0.8 g HEPES per 100 ml plasma. Mix and allow HEPES to dissolve (about 15 to 20 minutes). Fill stainless steel lyophilization trays with empty vials. Dispense exact 0.5 ml aliquots in each vial. Place rubber bung in each vial to depth of narrow ridge on bung. Ensure air access out of vial. Freeze at -70 C for a minimum of three hours. Freeze at -70 C for a minimum of three hours. Turn on freeze-dryer unit. Activate fridge unit. Place trays in shelving unit (up to eight). Place shelving unit in clear plastic chamber over air exit port on top of freeze-dryer. Activate pump. Ensure that the above two steps and this step are completed within three to four minutes to prevent plasma thaw commencing. If partial thaw occurs, material will froth and freeze dry poorly, and it must be discarded. Confirm that vacuum is developing by movement in pressure gauge (visible movement within a few minutes) and by immobility of plasma chamber under light lateral manual pressure. Leave under vacuum for five days. Seal vials under vacuum by screwing down handles on shelving unit. Allow air access through entry port. Defrost and dry freeze-dryer. Store lyophilized plasmas at -20 C, and cap as soon as possible. To confirm even lyophilization of plasma, test four to six vials selected from different locations within the steel trays. Determine PT and APTT, which should not vary by more than 6% 8%. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2228

Figure.4. Blood plasma freeze dryer Types of Freeze Dryers 8 There are essentially three categories of freeze-driers: the rotary evaporator freeze-drier, the manifold freeze-drier, and the tray freeze-drier. 1. Rotary freeze-driers are usually used with liquid products, such as pharmaceutical solutions and tissue extracts. Rotary freeze-dryers are usually used for drying pellets, cubes and other pourable substances. The rotary dryers have a cylindrical reservoir that is rotated during drying to achieve a more uniform drying throughout the substance. Tray style freeze-dryers usually have rectangular reservoir with shelves on which products, such as pharmaceutical solutions and tissue extracts, can be placed in trays, vials and other containers. Figure.5. Rotary freeze dryer. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2229

2. Manifold freeze-driers are usually used when drying a large amount of small containers and the product will be used in a short period of time. A manifold drier will dry the product to less than 5% moisture content. Without heat, only primary drying (removal of the unbound water) can be achieved. A heater must be added for secondary drying, which will remove the bound water and will produce a lower moisture content. Figure.6. Benchtop K Manifold freeze dryer 3. Tray freeze-driers are more sophisticated and are used to dry a variety of materials. A tray freeze-drier is used to produce the driest product for long-term storage. A tray freeze-drier allows the product to be frozen in place and performs both primary (unbound water removal) and secondary (bound water removal) freeze-drying, thus producing the driest possible end-product. Tray freeze-drierdrier is supplied with a stoppering mechanism that allows a stopper to be pressed into place, sealing the vial before it is can dry products in bulk or in vials. When drying in vials, the freeze- exposed to the atmosphere. This is used for long-term storage, such as vaccines. Figure.7. Tray freeze dryer IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2230

Improved Freeze Drying Techniques Improved freeze drying techniques are being developed to extend the range of products that can be freeze dried, to improve the quality of the product, and to produce the product faster with less labor. Ever since the 1930s, industrial freeze drying had been dependent on a single type of equipment: the tray freeze drier. In 2005 a quicker and less-labor intensive freeze drying method was developed for bulk materials. This freeze drying process proved to be able to produce free-flowing powder from a single vessel. Known as [Active Freeze Drying] AFD technology, the new process used continuous motion to improve mass transfer and hence cutting processing time, while also eliminating the need to transfer to and from drying trays and downstream size reduction devices. Figure.8. Development freeze dryer Sterile Production Grade Freeze Dryers Freeze Dryers used for processing pharmaceutical and viral products require sterilization between freeze drying runs. The most common form of sterilization is steam sterilization. Not only do steam sterilizable freeze dryers have to be IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2231

capable of maintaining a full vacuum they also need to be capable of maintaining high temperature and high pressures, which requires special design consideration and construction. Figure.5.. sterile production grade freeze dryers MILLROCK TECHNOLOGIES Freeze Dryers used for Biotech and Industrial applications can vary greatly. To accommodate the large range of requirements, Millrock Technology offers systems in either 304 stainless steel for non-pharmaceutical applications and 316L stainless steel for pharmaceutical applications. Condenser assemblies can be internal or external with either bulk or stoppering style shelf assemblies. Cylindrical chambers offer cost savings and can be supplied up to 80 square feet in shelf area. Features such as isolation valves and clean-in-place (CIP) piping can be provided. Millrock Technology works with each customer to fit their specific application needs. Selecting the right components and the right controls is critical to a successful freeze dryer project. 1. Research series freeze dryer Fifure.9. Research series freeze dryer IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2232

Shelf Area from 2 Sq ft to 10 Sq ft Full PC/PLC Control Standard High speed Ethernet connection Remote network access Redundant data logging with high speed data acquisition -53C or -85C Condenser Temperature 30L Condenser Capacity Remote Condenser with Hot Gas Defrost From 1 to 5 Shelves in Bulk or Stoppering -45C or -70C Shelf Temperature Fluid Filled Hollow Shelves, 12"x 24" Each Rectangular 316L Chamber 10PSI Hydraulic Stoppering Pirani Pressure Control and Gas Backfill Standard New! Opti-Dry Auto Run optimization software option New! Hydrogen Peroxide Sterilization Isolator Interface Option The Millrock Research Series is a new generation laboratory research shelf freeze dryer. Product can be loaded on the shelves and frozen prior to drying. A rectangular chamber offers a compact profile with either stoppering or bulk assembly. Hydraulic stoppering is provided by a bottom up hydraulic cylinder. Both options have a radiant heat plate above the top shelf for uniform drying on all shelves. All internal surfaces are constructed of 316L stainless steel and offer a polish of 20 Ra or better. A coil condenser is located between the product and the vacuum pump providing maximum condensing surface area and vacuum pump System Specifications protection. A 4" vapor port connects the two chambers, with a 6" option. An IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2233

isolation valve can be added for end of drying testing. Hot gas is used for defrosting the condenser when drying is complete. Fluid filled hollow shelves with a serpentinee fluid path provide the most uniform shelf temperature possible resulting in consistent and repeatable drying. All units come with one product tray per shelf, either a removable bottom or bulk. Options include a Shelf Latching Kit, Isolation Valve, Capacitance Manometer, Moisture Sensor, Clean Room Configuration, Sample Extractor, 6" vapor ports, and much more. 2. Max series freeze dryer Figure.10. max series freeze dryer Shelf Area from 10 Sq ft to 20 Sq ft 5 to 10 Shelves in Bulk or Stoppering Full PC/PLC Control Standard -53C or 85C Condenser Temperature 30L Condenser Capacity Remote Condenser with Hot Gas Defrost -45C or -70C Shelf Temperature Fluid Filled Hollow Shelves, 12 x 24 Each Rectangular 316L Chamber 10PSI Hydraulic Stoppering IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2234

Pirani Pressure Control Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology Clean Room Configuration Available New! Hydrogen Peroxide Sterilization Isolator Interface Option The Max Series Freeze Dryer provides the maximum shelf space possible from phase one research to clinical production and small volume production. A rectangular chamber offers a compact profile with either stoppering or bulk assembly. Hydraulic stoppering is provided by a bottom up hydraulic cylinder. Both options have a radiant heat plate above the top shelf for uniform drying on all shelves. All internal surfaces are constructed of 316L stainless steel and offer a polish of 20 Ra or better. A coil condenser is located between the product and the vacuum pump providing maximum condensing surface area and vacuum pump protection.with the ability to add an optional isolation valve, a 6" vapor port connects the two chambers. An isolation valve can be used for end of drying testing. Hot gas is used for defrosting the condenser when drying is complete. Fluid filled hollow shelves with a serpentinee fluid path provide the most uniform shelf temperature possible and results in consistent and repeatable drying. All units come with one product tray per shelf, either a removable bottom or bulk. Options include a Shelf Latching Kit, Isolation Valve, Capacitance Manometer, Moisture Sensor, Clean Room Configuration, Sample Extractor, 6"/8" vapor ports, and much more. 3. Laboratory series freeze dryers Figure.11. laboratory series freeze dryers IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2235

Shelf Area 3.75 sq ft Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology Shelf and Manifold Dryer PC/PLC Control System -53C or 85C Condenser Temperature -45C or 70C Shelf Temperature 6L in 24 hours, 8L total capacity Rectangular 316L Chamber 3 Shelf Bulk or 3 Shelf Stoppering 10 x 18 Shelves Fluid Filled Hollow Shelves Internal Condenser with Hot Gas Defrost Pneumatic stoppering (6PSI) CFC-free Refrigeration In a class by itself, the Millrock Laboratory Series is a new generation freeze dryer that is both a shelf and manifold dryer. Product can be loaded on the shelves and frozen prior to drying or can be frozen in an optional shell freezer for manifold drying. A rectangular chamber offers a compact profile with either a three shelf stoppering or three shelf bulk assembly. Both options have a radiant heat plate above the top shelf for uniform drying on all shelves. All internal surfaces are constructed of 316L stainless steel and offer a polish of 20 Ra or better. For the fastest drying rates and maximum pump protection the condenser is located inside the chamber beneath the bottom shelf. Hot gas is used for defrosting the condenser when drying is complete. Fluid filled hollow shelves with a serpentine fluid path provide the most uniform shelf temperature possible and results in consistent and repeatable drying. All units come with one product tray per shelf, either a removable bottom or bulk. Pneumatic stoppering is provided by a bottom up pneumatic cylinder, which is powered by a self-contained air pump. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2236

4. Console manifold freeze dryer Figure.12. console manifold freeze dryer Advanced Microprocessor Controls Manifold Dryer with 24 to 48 ports 20 Liters in 24 hours, 30L Capacity Internal Condensing Coil 316L -53C or 85C Condenser Temperature 316L Construction CFC-free Refrigeration Hot Gas Defrost 200 LPM Vacuum Pump Millrock has responded to customer demandd for a cost effective high performance manifold freeze dryer. The Manifold Series takes a market proven design and has added the most advanced microprocessor control available on a manifold unit. The Manifold Freeze-Dryer Series boasts a 20L per 24 hour condensing rate with a 30L capacity. For water based samples, the 53 C condenser is the perfect choice. For samples containing solvents or with low eutectic points, the 85 C condenser provides maximum performance. For labs that need quick turn-around, hot gas defrost supplies fast/automatic defrosting. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2237

Choose from a 24 or 48 port manifold. All provide ample space for small and large samples to be dried simultaneously. Straight-thru vacuum valves offer a more direct vapor path. Advances in Lyophilisation 1. Alaska ln2 for freeze-drying 9 : Cryogenic heat exchanger Figure.10. Freeze-drying cycle optimization using cryogenic cooling and ALASKA range heat exchangers. ALASKA LN2 for freeze-drying: an alternative to using conventional coolants (CFC, HCFC) which contribute to the destruction of the ozone layer. Liquid nitrogen has a higher cooling capacity and makes it possible to improve freeze dryer performance, by reducing cycle times. ALASKA LN2 for freeze-drying is the Air Liquide solution for providing the cooling capacity required by a freeze dryer. Operating principle: Liquid nitrogen is stored in liquid form at a temperature of -196 C. It is injected - into the ALASKA exchanger to cool silicone oil to the desired freezing temperature, typically 40 C. - into the winding to trap water vapor in the form of ice. The trap temperature can vary between -60 C and -80 C. The gaseous nitrogen is returned to the atmosphere via a vent A simple and reliable system: 5 components forming the system: The LN2 tank The vacuum-insulated LN2 transfer line The ALASKA exchanger IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2238

The trap windings Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology The temperature regulation system and the LN2 operating tools supplied with the ALASKA exchanger An economical solution: - System reliability due to absence of rotating machines - Maintenance and operating costs - Freeze-dried product quality - Productivity gain due to cycle time reduction ALASKA LN2 is a suitable and economical solution, compared to conventional mechanical cooling systems Range An exchanger range from 5 to 100 kw with the possibility to manufacture higher-capacity equipment if required. Table.1 Exchanger CF 5 CF 12,5 CF 25 CF 37,5 CF 50 CF 75 CF 100 Nominal cooling power (kw) 5 12,5 25 37,5 50 75 100 Key benefits Comparison with mechanical cooling units: ALASKA LN2 offers the following advantages: Lower maintenance and no specific expertise required. Compact size, no noise pollution. Suppression of use of gases that destroy the ozone layer. No regulatory constraints Process Easy operation due to the flexibility of the system. Greater reliability and reproducibility of freeze-drying cycles. Greater cooling capacity than conventional systems, which is constant throughout the temperature range and instantaneously available. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2239

Rapolu Bharath kumar kumar** et al. /International Journal Of Pharmacy&Technology Cycle time reduction. Possibility to reach very low temperatures for specific recipes Low operating cost No electricity No backup generator No cooling water Lower maintenance costs Lower number of shutdowns due to failure. Main technical characteristics Specific liquid nitrogen exchangers. Large heat exchange surface area Insulation making it possible to limit heat losses LN2 consumption optimization Sturdy stainless steel design "Ready-to-start" skid assembly Figure.13. ALASKA LN2 FREEZE DRYER IJPT Oct-2012 Vol. 4 Issue No.3 2215--2243 Page 2240

2. Sharp freezer 10 Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology The Sharp Freeze is a flexible and compact laboratory freeze-dryer. The system's performance and reliability ensure that a constant low temperature of up to -110C (depending on the model) is maintained. Fitted with a highly insulated chamber, the Sharp Freeze efficiently traps all vapors and prevents them from escaping, ensuring a longer working life for the vacuum system attached.the Sharp Freeze -110C has dual compressors for greater efficiency and includes a step start feature to preserve the life of the compressors and to maintain a constant temperature at the various stages of operation. An optional Pirani gauge provides precise digital pressure readout for controlling the freeze drying process. Utilizing the option RS232C port, the condenser temperature can be automatically monitored and recorded at all times. A generous 4 liter chamber as standard, constructed of 316 stainless steel, allows the Sharp Freeze to suit most laboratory applications, but custom models are available for more challenging applications. A drain valve allows fast cleaning and easy emptying of the condenser, and a special glass trap is available for trapping highly corrosive chemicals. Key Features Produces lower vacuum pressure Increased efficiency of lyophilization Increased working life of pump and vacuum system Energy efficient Key Specifications Ultimate temperature -55C or -110C Total Trap Volume - 4 liters Cooling Capacity; water up to 2 kg/hour Condenser Material; 316 Stainless Steel Digital temperature display Microprocessor control Overload and malfunction alarms Compressor start delay IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2241

Figure.14. Sharp freezer Factors Effecting Formulation The active constitution of many pharmaceutical products is present in such a small quantity that if freeze dried alone its presence would be hard to predict it visually. The solid components of the orginal product should be between 5% to 30%. Therefore exipients are added to increase the amount of solids. Such exipients are called bulking agents, most commonly used bulking agents is mannitol. Buffering agents such as sodium or potassium phosphate, sodium citrate and sodium acetate are added to buffer the product or to protect the product from freeze drying. Sucrose, trehalose, dextran and amino acids are commonly used lyoprotectants. Conclusion Conventional drying methods also have a major disadvantage as the high temperatures used can cause chemical or physical changes to the product. For pharmaceuticals and bio-products, this would cause a reduction in biological activity, which could render them ineffective. With freeze drying, delicate, unstable or heat-sensitive drugs and biologicals can be dried at low temperatures without damaging their physical structure. Freeze-dried products can be reconstituted quickly and easily, which is particularly valuable in the case of emergency vaccines and antibodies, which need to be administered as quickly as possible. IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2242

References: Rapolu Bharath kumar* et al. /International Journal Of Pharmacy&Technology 1. 1.http://www.rpi.edhttp://www.scribd.com/doc/377243/Lyophilization-Basicsu/dept/chem-eng/Biotech- Environ/LYO/definition.html 2. http://www.freezedrying.com/freeze_drying_principles.html 3. http://en.wikipedia.org/wiki/freeze-drying 4. http://www.pharmafocusasia.com/manufacturing/lyophilisation.htm 5. http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=7&cad=rja&ved=0cf 0QFjAG&url=http%3A%2F%2Fwww.champa.kku.ac.th%2Fthanaset%2FLyophilization.doc&ei=9E5AUJXA C8rPrQfSoYH4DA&usg=AFQjCNGfialqCMgKqvf35BRbOaicogg1Bw 6..http://www.rpi.edu/dept/chem-eng/Biotech-Environ/DOWNSTREAM/sect3.htm. 7. http://www1.wfh.org/2/docs/publications/diagnosis_and_treatment/lab_manual2010/lab_manual_section- 40.pdf 8. http://www.millrocktech.com/ 9. http://www.airliquideadvancedtechnologies.com/en/our-offer/chemicals pharmaceuticals/temperatureregulation/alaska-ln2-en-lyophilisation-echangeur-de-chaleur-cryogenique.html 10. http://www.aapptec.com/view_p.aspx?id=44. 11. http://www.rpi.edu/dept/chem-eng/biotech-environ/downstream/fig4.htm 12..http://www.scribd.com/doc/377243/Lyophilization-Basics 13. http://www.labmanager.com/?articles.view/articleno/6127/ 14. http://www.iptonline.com/articles/public/bocedwardspharmaceuticalsystems.pdf Corresponding Author: Rapolu Bharath kumar*, Email:bharath.rapolu@gmail.com IJPT Oct-2012 Vol. 4 Issue No.3 2215-2243 Page 2243