INTRODUCTION:

The lyophilization means to make solvent loving. Operationally we could define freeze-drying as a controllable method of dehydrating labile products by vacuum desiccation. Technically, freeze-drying may be defined as:

· Freezing of the liquid sample, followed by the conversion of water into ice and crystallization of crystallizable solutes as well as formation of an amorphous matrix comprising non crystallizing solutes associated with unfrozen moisture.

· Sublimation of freezed ice under vacuum. Evaporation of moisture from the amorphous matrix.

· Desorption of chemisorbed water resident in the apparently dried cake.^[1]

Lyophilization or freeze drying is a process in which water is frozen, and removal from the sample, initially by sublimation (primary drying) and then by desorption (secondary drying). Freeze drying is a process of drying in which water is sublimed from the product after it is frozen for prolonged storage drying process applicable to manufacture of certain chemicals, pharmaceuticals and biologicals that are thermolabile or otherwise unstable in aqueous solution sand. it is stable in the dry state. The term lyophilization describes a process to produce a product that loves thedry state. Lyophilization is performed at temperature and pressure conditions below the triple point of water, to enable sublimation of ice.^[2]The term “lyophilization” describes a process to produce a product that „ loves the dry state.‟ However, this term does not include the freezing process. Therefore, although lyophilization and freeze drying are used interchangeably, freeze drying is a more descriptive term. Lyophilization is the most common method for manufacturing parentrals when aqueous solution stability is an issue. It is central to the protection of materials, which require low moisture content (less than 1%) in order to ensure stability and require a sterile and gentle preservation process.

PRINCIPLE:

The main principle involved in lyophilisation a phenomenon called sublimation, where water passes directly from solid state (ice) to the vapor state without passing through the Sublimation of water can take place at pressures and temperature below triple point i.e. 4.579 mm of Hg and 0.0099 degree Celsius.^[3]The material to be dried is first frozen and then subjected under ahigh vacuum to heat (by conduction or radiation or by both) so that frozen liquid sublimes leaving only solid,dried components of the original liquid. The concentration gradient of water vapor between the drying front and condenser is the driving force for removal of water during lyophilization.^[4]

To extract water from formulation, the process of lyophilization consists of :

1. Freezing the formulation so that the water in the food become ice.

2. Under a vacuum, sublimating the ice directly into water vapour.

3. Drawing off the water vapour.

4. Once the ice is sublimated,the foods are freeze dried and can be removed from the machine.^[5]

The principle of lyophilization is as follows.

1. Heat transfer:

Heat supplies the energy necessary for sublimation of the water. An ice crystal is composed of pure water called as crystal lattice. The many molecules have contain natural vibrations, so that extra thermal energy increases and probability of water molecules breaking free. When the water molecules breaks free, it diffuses through the dried surface of the solid and sublime, the outer surface of the specimen thickened, and thus more energy is required to transport the molecules through the dry shell. Heat transfer to the product can be divided into three components: direct conduction, gas conduction and radiation. The pathways for transfer of energy through these three mechanisms are illustrated in fig 1.

Conduction is the main contributor to the heat transfer. It represents the heat energy transmitted from the shelf to the vial at the area where both are in direct contact. It is depends on the container type used, is especially low for well plates or molded vials, and only covers a fraction of the total vial bottom even for tubular vials designed for lyophilization. The amount of heat conveyed is proportional to the temperature difference between the cold vial and the warmer shelf. The driving force in conduction is the temperature gradient between different solids. Conduction can be modeled by Fourier's law: 1

dQ/dt=A λ dT/dZ …….1

Where,

dQ/dt - Heat flow,

A - Area of the surface,

λ - Thermal conductivity of the material and

dT - Temperature gradient across the thickness of the material.

Figure 1: Type of heat transfer to the products.

For solids in series, the heat transfer rate, dQ/dt, can be thought of as the temperature gradient divided by the sum of the resistances. The resistances to heat transfer are shown in Fig 2. Heat is supplied to the interior of the shelf, either through electric coils or by a heated flowing liquid. The first resistance is the shelf, with a temperature difference from the interior to the surface. The next resistance is the tray or pan upon which the vials are placed, with a temperature difference from the shelf surface to the top of the tray. The third resistance is the glass vial, with a temperature difference between the tray surface and the bottom of the product in the vial. The fourth resistance is the frozen product inside the vial, with a temperature gradient between the ice at the bottom of the vial and the ice at the sublimation interface.

Radiation heat transfer must also be taken into account in lyophilization. Heat transfer by radiation takes place between two surfaces with different temperatures, i.e. the cold vial and the shelf, the shelf, as well as door and walls of chamber. The warmer surface radiates electromagnetic energy which is absorbed by the colder surface. Although this pathway also depends on the distance between the surfaces and temperature difference. Stefan Boltzmann equation describe radiative heat transfer as follows

dQr/dt = Avēσ(T₂⁴-T₁⁴)……….2

Where,

dQr/dt -Represents the amount of energy per time transmitted by radiation,

Av - Vial area (top or bottom5),

ē - Effective emissivity for exchange of radiation (between 0 and 1),

σ - Boltzmann constant, and

(T₂⁴ —T₁⁴ ) - Difference between the temperature of the two surfaces to the 4th power.

The effective emissivity is an important parameter for surface materials used in the construction of a freeze dryer. While acrylic glass shows especially high emissivity (0.95), the radiation of polished stainless steel is much lower (0.4). This difference needs to be in lyophilization during transfer and scale-up of lyophilization cycles between freeze-dryers with different radiation characteristics.

2. Mass transfer:

The mass transfer of water vapor from the product to the condenser is determined by several resistances to vapor flow that limit the flow rate. The most important factor is the resistance of the already dried layer to mass transfer, the so called product resistance (Rp). The water vapor which sublimes at the sublimation front needs to diffuse through a network of small pores in the dried matrix. These pores are created when ice crystals are removed by sublimation, and their size, shape and interconnection are influenced by the freezing process. Rp values depend on the thickness of the already dried cake layer, and change during the course of the drying process.

Figure 2: Resistances and their relative contributions in mass transfer

In modeling, the product can be thought of a porous solid, with Knudson flow. The stopper can be modeled as a solid with transition flow through small tubes. The chamber can be modeled as a gas with viscous flow. The resistance associated with the product, Rp, depends on the cross sectional area of the product, Ap by However, this really becomes a movingboundary problem, as Rp increases with time as the ice moves out of the product cake and must be solved through numerical methods.

2.1 Coupling between heat and mass transfer:

During the steady state of primary drying, the amount of heat introduced in product is equilibrium with amount of heat removed in sublimation of ice. During freeze-drying Heat and mass transfer are coupled which can be described by:

dQ/dt=dm/dt.ΔHS+ms.cv(dT/dt)……3

Where,

dQ/dt-Flow to heat in to product,

dm/dt - Removal mass by sublimation,

ΔHS - Temperature-dependent heat of sublimation of ice (call/g), ms - Sample mass (g),

cv - Specific heat of the sample (call/K*g) and

dT/dt - Change of product temperature (K/s).

The first term describes the rate of heat removal by sublimation, the next term signifies the rate of heat removal through a change in product temperature which is mainly the case during the early stage of primary drying. Since the second specific heat term is usually small compared to the sublimation term, the heat transfer during steady state primary drying can be described with the simplified equation: .

dQ/dt= dm/dt.ΔHS……..4

This implies that essentially all heat introduced into the product is used to convert ice into water vapor by sublimation, and the product temperature is assumed to remain constant. This simplified model is the basis for numerous modeling approaches of the freeze-drying process.^[6]

Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, passing the ice to change directly from solid to vapor without passing through a liquid phase.^[7]Lyophilization is done at temperature and pressure conditions below the triple point, to allow sublimation of ice. The complete process is performed at low temperature and pressure, hence is appropriate for drying of thermolabile compounds. Steps involved in lyophilization start from sample preparation followed by low temperature freezing, high vacuum primary drying and increasing temperature secondary drying, to obtain the final dried product with anticipated moisture content.^[8]The driving force for removal of water during lyophilization is concentration gradients of water vapor between drying front and condenser. During primary drying process an increases the temperature.

vapor pressure of water increases. Therefore, primary drying temperature should be below the critical process temperature or as per possible high, to avoid a loss of cake structure. In amorphous substance, or eutectic melt for the crystalline substance the critical process temperature is collapse temperature. During freezing, ice crystals start separating out until the solution becomes maximally concentrated. On further cooling, phase separation of the solute and ice takes place.^[9]

Reasons for Lyophilization:

· Material chemically unstable in solution

· Low temperature drying process

· Compatible with protein pharmaceuticals

· The amorphous form of the drug is desirable (i.e., solubility)

· Low particulate contamination

· Compatible with aseptic/sterile processing.^[10]

Importance of Lyophilization process:

Lyophilized Product characteristics

· Long stability.

· Minimum reconstitution time.

· Elegant cake appearance.

· Maintain original dosage form characteristics upon reconstitution, including solution properties; structure and conformation of proteins; and particle-size dispersion in suspensions.

· After reconstitution isotonicity maintained.^[11]

Desirable freeze drying characterstics:

1 Uniform color of products.

2 Sufficient drying of products.

3 Sufficient porosity of finally dried products.

4 Chemical stability of products.

5 Intact cake of products.

6 Sufficient strength in terms of assay pH

ADVANTAGES:

· Oxidizable substances are well protected under vacuum conditions.

· Long preservation period owing to 95% 99.5% water removal. Loading quantity accurate and content uniform.

· Little contamination owing to aseptic process.

· Minimal loss in volatile chemicals and heat sensitive nutrient and fragrant components.

· Minimal changes in the properties because microbe growth and enzyme effect can not be exerted under low temperature.

· Transportation and storage under normal temperature.

· Rapid reconstitution time.

· Constituents of the dried material remain homogenously dispersed.

· Product is process in the liquid form.

· Sterility of product can be achieved and maintained.

DISADVANTAGES:

· Volatile compounds may be removed by high vacuum.

· Single most expensive unit operation.

· Stability problems associated with individual drugs.

· Some issues associated with sterilization and sterility assurance of the dryer chamber and aseptic loading of vials into the chamber.^[12]

LYOPHILIZATION / FREEZE DRYING PROCESS:

Freeze drying is mainly used to remove the water from sensitive products, mostly of biological origin, without damaging them, so they can be preserved easily, in a permanently storable state and be reconstituted simply by adding water [13]. Examples of freeze dried products are: antibiotics, bacteria, sera, vaccines, diagnostic medications, protein containing and biotechnological products, cells and tissues, and chemicals. The product to be dried is frozen under atmospheric pressure. Then, in an initial drying phase referred to as primary drying, the water (in form of ice) is removed by sublimation; in the second phase, called secondary drying, it is removed by desorption. Freeze drying is carried out under vacuum.^[14]

The freeze drying process consists of three stages:

1 Freezing,

2 Primary drying, and

3 Secondary drying.

FREEZING:

Since freeze drying is a change in state from the solid phase to the gaseous phase, material to be freeze dried must first be adequately prefrozen. The method of freezing and the final temperature of the frozen product can affect the ability to successfully freeze dry the material. Rapid cooling results in small ice crystals, use fulin preserving structures to be examined microscopically, but resulting in a product that is more difficult to freeze dry. Slower cooling results in larger ice crystals and less restrictive channels in the matrix during the drying process Products freeze in two ways, depending on the makeup of the product. The majority of products that are subjected to freeze drying consist primarily of water, the solvent, and the materials dissolved or suspended in the water.^[15]

Primarydrying:

Low product temperature and the corresponding low vapor pressure of ice result in extensive primary drying times. It has been reported that elevation of product temperature by 1°C can reduce the overall primary drying time by as much as 13%, which offers enormous potential of saving process time and manufacturing costs when administering more aggressive product temperatures.^[16]However, an increase of product temperatures to temperatures above the “critical formulation temperature” which refers to the eutectic melting temperature, TE, for crystalline and to Tc orTg for amorphous materials, mostly leads to loss of cake structure. If the critical temperature is exceeded, the dried pore structure close to the sublimation front that still contains high amounts of water can undergo viscous flow, resulting in fusion of pores and formation of holes in the cake structure. This occurrence is associated with a reduction of inner surface area as well as elevated moisture contents with potentially detrimental effects on reconstitution time and completeness as well as API stability.^[17]Most importantly, the cake shows shrinkage or may fully collapse, making the product unsuitable for sale and application in patients due to the lack of elegance. The critical formulation temperature can be determined using Freeze Dry Microscopy (FDM) which allows observation of the drying cake structure under vacuum at varying temperatures.^[18]Once the collapse temperature is reached it is possible to observe formation of holes in the dried cake structure. Since the sample is being dried during the experiment, the conditions are more similar to lyophilization than alternative methods, making the results more representative for a vial freeze drying process.^[19]A different approach to determine the critical formulation temperature is Differential Scanning Calorimetry (DSC) which measures the heat flow and thermal properties of the frozen sample. This way it is possible to determine the glass transition temperature of the maximally freeze concentrated solute, Tg, which is indicative for molecular mobility in the amorphous matrix.^[20]Since no removal of Water is involved, the critical temperature is not as representative for vial freeze drying as the collapse temperature determined using FDM. It is possible to increase the critical temperature by crystallizing salts (i.e. buffers etc.) quantitatively during freezing, or by adding amorphous excipients with high Tg‟ values such as dextran or cyclodextrines.^[21]

Fig 3

SECONDARY DRYING:

After primary freeze drying is complete, and all ice has sublimed, bound moisture is still present in the product. The product appears dry, but the residual moisture content may be as high as 7-8% continued drying is necessary at warmer temperature to reduce the residual moisture content to optimum values. This process is called„ Isothermal Desorption‟ as the bound water is desorbed from the product.^[22]Secondary drying is normally continued at a product temperature higher than ambient but compatible with the sensitivity of the product. In contrast to processing conditions for primary drying which use low shelf temperature and a moderate vacuum, desorption drying is facilitated by raising shelf temperature and reducing chamber pressure to a minimum. Care should be exercised in raising shelf temperature too highly; since, protein polymerization or biodegradation may result from using high processing temperature during secondary drying. Secondary drying is usually carried out for approximately 1/3 or 1/2 the time required for primary drying. The general practice in freeze drying is to increase the shelf temperature during secondary drying and to decrease chamber pressure to the lowest attainable level. The practice is based on the ice is no longer present and there is no concern about “melt track” the product can withstand higher heat input.^[23]Also, the water remaining during secondary drying is more strongly bound, thus requiring more energy for its removal. Decreasing the chamber pressure to the maximum attainable vacuum has traditionally been thought to favor desorption of water.

The Importance of stability:

The main consideration in lyophilization of protein formulations is long-term stability, which is related to water content, says Enric Jo, director and plant manager of ReigJofre, a European development and manufacturing group serving the pharmaceutical market. “For small-molecule drugs, it is possible in most cases to formulate them without excipients, or just by adding a bulking agent or pH modifier, to obtain a liquid formulation that is stable enough to endure the necessary duration spent before it is freeze-dried. The moisture content in the products is usually sufficiently low to ensure that the formulation remains stable for long periods.” For proteins, however, the situation is more complicated because proteins are labile molecules. Their stability is related to the water content of the formulation, but at the same time, the active form of a protein is related to the conformational structure that needs some water content to avoid denaturation processes. According to Jo, these stability issues can be avoided through formulation optimization and adequate process control. A new stability concept can be described as thermodynamic stability. This stability refers to the position of equilibrium between native and unfolded conformations,” says Jo. “The problem is further complicated because while a protein may exhibit thermodynamic instability during freeze drying and unfold, if no irreversible reactions (e.g., aggregation) occur during storage or during reconstitution, the reconstituted protein may refold completely within seconds and display perfect pharmaceutical stability.^[24]

Use of Cryoprotectant:

Acryoprotectantis a substance used to protect biological tissue from freezing damage (i.e. that dueto ice formation). Arctic and Antarctic insects, fish and amphibians create cryoprotectants (anti freeze compounds and anti freeze proteins) in their bodies to minimize freezing damage during cold winter periods. Cryoprotectants are also used to preserve living materials in the study of biology and to preserve food products. The most popular cryoprotectants encountered in the literature for freeze-drying microparticles are sugars: trehalose, sucrose, glucose and mannitol. These sugars are known to vitrify at a specific temperature denoted Tg.^[25,26]The immobilization of micro particles within a glassy matrix of cryoprotectant can prevent their aggregation and protect them against the mechanical stress of ice crystals. Generally, freezing must be carried out below Tgof a frozen amorphous sample or below Teu (eutectic crystallization temperature) which is the crystallization temperature of soluble component as a mixture with ice, if it is in a crystalline state in order to ensure the total solidification of the sample.^[27]

EXCIPIENTS USED IN LYOPHILIZED FORMULATION:

The design of aqueous lyophilized formulation is dependent on the requirements of the active pharmaceutical ingredient (API) and intended route of administration. A may consist of one or more excipients that perform one or more functions. Excipients may be characterized as buffers and pH adjusters, bulking agents, stabilizers, and tonicity modifiers.^[28]

Buffers:

Buffers are required in pharmaceutical formulations to stabilize pH. In the development of lyophilized formulations, the choice of buffer can be critical. Phosphate buffers, especially sodium phosphate, undergo drastic pH changes during freezing. A good approach is to use low concentrations of a buffer that under goes minimal pH change during freezing such as citrate and histidine buffers.

Bulking agents:

The purpose of the bulking agent is to provide bulk to the formulation. This is important in cases in which very low concentrations of the active ingredient are used. Crystalline bulking agents produce an elegant cake structure with good mechanical properties. However, these materials often are in effective in stabilizing products such as emulsions, proteins and liposomes but may be suitable for small chemical drugs and some peptides. If a crystalline phase is suitable, mannitol can be used. Sucrose or one of the other disaccharides can be used in a protein or liposome product.

Stabilizers:

In addition to being bulking agents, disaccharides form an amorphous sugar glass and have proven to be most effective in stabilizing products such as liposomes and proteins during lyophilization. Sucrose and trehalose are inert and have been used in stabilizing liposome, protein, and virus formulations. Glucose, lactose, and maltose are reducing sugars and can bereduce proteins by means of the mallard reaction.

Tonicity adjusters:

In several cases, an isotonic formulation might be required. The need for such a formulation may be dictated by either the stability requirements of the bulk solution or those for the route of administration. Excipients such as mannitol, sucrose, glycine, glycerol, and sodium chloride are good tonicity adjusters. Glycine can lower the glass transition temperature if it is maintained inthe amorphous phase. Tonicity modifiers also can be included diluents rather than the formulation.

The lyophilization equipment:

The environmental conditions necessary for the lyophilization process, sub ambient temperatures and sub-atmospheric pressures, are achieved by the lyophilization equipment. The following gives a general description of the essential components and their function in a lyophilize. The general design of common lyophilizer is displayed in Figure :-

Figure 4: Lyophilizer Design

Essential Components Chamber:

This is the vacuum tight box, sometimes called the lyophilization chamber or cabinet. The chamber contains shelf or shelves for processing product. The chamber can also fit with a stoppering system. It is typically made of stainless steel and usually highly polished on the inside and insulated and clad on the outside.^[29]The door locking arrangement by a hydraulic or electric motor.

Shelves:

A small research freeze dryer may have only one shelf but all others will have several. The shelf design is made more complicated because of the several functions it has to perform. The shelves will be connected to the silicone oil system through either fixed or flexible hoses. Shelves can be manufactured in sizes up to 4 m2 in area.^[30]The process condenser is sometimes referred as just the condenser or the cold trap. It is designed to trap the solvent, which is usually water, during the drying process. The process condenser will consist of coils or sometimes plates which are refrigerated to allow temperature. These refrigerated coils or plates may be in a vessel separate to the chamber, or they could be located within the same chamber as the shelves.

Shelf fluid system:

The freeze-drying process requires that the product is first frozen and then energy in the form of heat is applied throughout the drying phases of the cycle. This energy exchange is traditionally done by circulating a fluid through the shelves at a desired temperature.^[31] The temperature is set in an external heat exchange system consisting of cooling heat exchangers and an electrical heater. The fluid circulated is normally silicone oil. This will be pumped around the circuit at a low pressure in a sealed circuit by means of a pump.

Refrigeration system:

The product to be freeze dried is either frozen before into the dryer or frozen whilst on the shelves. A considerable amount of energy is needed to this duty. Compressors or sometimes liquid nitrogen supplies the cooling energy. Most often multiply compressors are needed and the compressor may perform two duties, one to cool the shelves and the second to cool the process condenser.

Vacuum system:

To remove solvent in a reasonable time, vacuum must be applied during the drying process. The vacuum level required will be typically in the range of 50 to 100μ bar. To achieve such a low vacuum, a two stage rotary vacuum pump is used. For large chambers, multiple pumps may be used.

APPLICATIONS:

Pharmaceutical and biotechnology:

Pharmaceutical companies often use freeze-drying to increase the shelf life of products, such as vaccines and other injectables.^[32]By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form for injection.

Food Industry:

Freeze-drying is used to preserve food and make it very light weight. The process has been popularized in the forms of freeze-dried ice cream, an example of astronaut food.

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 bioseparations, 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.^[33]

CONCLUSION:

Lyophilization provides a method of drying temperature labile materials. The freeze drying process is divided into 3 steps: Freezing, Primary Drying, Secondary Drying. Freeze drying is often the last choice in methods for drying materials, because the cost and time required. Changing the freezing, primary drying, or secondary drying. The lyophilization technique proved to be an advantage for development of stable injectable dosage form as the moisture content of the formulation is greatly reduced thus enhancing the stability of the product, ease of handling, rapid dissolution because of porous nature of the cake and easier transport of the material during shipping.About 50% of the currently biopharmaceuticals are lyophilized, representing the most common formulation strategy. In the freeze dried solid state, chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long term stability. The awareness of the complexity of the freezing process and its consequences on product quality and process performance is essential for successful lyophilization. The knowledge of how to control, or at least manipulate, the freezing step will help to develop more efficient lyophilization cycles and biopharmaceutical products with an improved stability

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Received on 16.11.2016 Accepted on 17.12.2016

Asian J. Res. Pharm. Sci. 2016; 6(4): 269-276.

DOI: 10.5958/2231-5659.2016.00038.2

ABSTRACT: