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Journal of Advanced Pharmaceutical Research. 2011, 2(4), 157-169.

Review paper Fundamentals and Applications of Lyophilization

Gannu Praveen Kumar*, Nooka Prashanth, Bairi Chaitanya Kumari Talla Padmavathi College of Pharmacy, Orus, Kareemabad, Warangal

Corresponding author E-mail: [email protected] Received: Oct 12, 2011; Accepted: Nov 23, 2011

ABSTRACT

Lyophilization of pharmaceutical solutions to produce an elegant stable powder has been a standard practice employed to manufacture of many marketed pharmaceutical injectable products. Lyophilization (freeze drying) is a process in which water is removed from a product, after which it is frozen and then placed under vacuum, followed by the ice changing directly from solid to vapor without passing through a liquid phase. Three unique and interdependent process consists they are processes freezing, primary drying (sublimation) and secondary drying (desorption) are detailed exhaustively in this review including it’s critical pharmaceutical applications

KEYWORDS: lyophilization, freezing, primary drying, secondary drying. 1. INTRODUCTION

Freeze drying or lyophilization in simple terms is a

dehydration technique in which an aqueous solution is

first frozen and subsequently dried by sublimation under

vacuum. The remaining solid undergoes additional drying

at elevated temperatures and forms a porous cake with

high internal surface area. By reconstituting the lyophile

with water for injection, it is easy to achieve a sterile,

particle free and accurately dosed solution that can be

directly administered parenterally. The aspect of the

freeze drying process that makes it different from other

dehydration techniques is that dehydration takes place

while the product is in a frozen state and under vacuum.

These conditions stabilize the product minimizing the

effects of oxidation and other degradation processes.

Substances that degrade in solution become the candidate

of freeze drying. It has become an accepted method of

processing heat sensitive products that require long term

storage at temperatures above freezing.

2. HISTORY

Freeze drying as a practical commercial process was

introduced during the time of Second World War and its

first application was found by Greaves in preservation of

blood plasma in the year 1944 (Greaves, 1954).

Production of freeze dried antibiotics mainly penicillin

with enhanced stability was achieved during the 1950‘s.

The biotech revolution in the 1990’s has lead to an

increasing demand for lyophilized products as well as

further investigation and optimization of the freeze drying

process. Costantino reported that 46% of the FDA

approved protein, peptide, vaccine, oligonucleotide and

cell-based products are produced by lyophilization

(Costantino and Pikal, 2004).

Typical biopharmaceutical products that are

manufactured by freeze drying are peptides and proteins

such as antibodies, enzymes or hormones. Other

important lyophilized pharmaceuticals are vaccines,

antibiotics and vitamins. The conservation of blood

plasma of rare blood groups is also an important area.

Besides pharmaceutical applications, lyophilization is

mainly used for stabilization of food products such as

coffee, herbs and fruits. Newly developed drugs often

show poor solubility and require novel dosage forms such

as liposomes, microparticles or nanoparticles to minimize

solubility problems and side effects due to toxicity. These

dosage forms are often inherently labile due to

agglomeration, and sedimentation. They can be stabilized

and manufactured by freeze drying. Other innovative

classes of drugs that can be produced by lyophilization

are DNA/RNA carriers or complexes.


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3. ADVANTAGES

Lyophilization has many advantages compared to other

drying and preserving techniques. It is a gentle drying

technique for sensitive products, they can be stored at

ambient temperature over a 2 year shelf life, enhanced

product stability in a dry state, easy reconstitution, greatly

reduces weight and makes the products easier to

transport, maintains food/biochemical and chemical

reagent quality, reconstitution of the dried product

facilitates use in emergency medicine and safe

application in hospitals, it is not limited to products for

parentral use, but can also be used for fast dissolving

sublingual tablets (Nail et al., 2002), Tablets can have

very low disintegration time and have great mouth feel

due to fast melting effect, it is much easier to achieve

sterility assurance and freedom of particles than using

other drying methods or handling of dry powders,

products sensitive to oxidation can be stoppered and

sealed within an inert atmosphere (i.e. nitrogen) to

minimize detrimental effects

4. DISADVANTAGES

Although lyophilization has many advantages compared

to other drying and preserving techniques it has quite a

few disadvantages. It is a long and cost intensive process,

requires sterile diluents for reconstitution, it should only

be used when product is unstable and heat-liable and the

limited amount of vials processed in each run restricts the

overall production capacity.

5. LYOPHILIZATION PROCESS

The lyophilization process consists of three stages:

5.1. Freezing

Freezing is a critical step in freeze drying process since

the micro structure formed during freezing determines

both the quality of the final product and its processing

characteristics, such as the rate of primary drying and

secondary drying. The product must be frozen to low

temperature to a point where it is completely solidified.

Freezing the product decreases chemical activity by

decreasing molecular movement. In general, freezing is

defined as the process of ice crystallization from super

cooled water. The freezing process first involves the

cooling of the solution until ice nucleation occurs. Ice

crystals begin to grow at a certain rate, resulting in freeze

concentration of the solution, a process that can result in

either crystalline or amorphous solids or in mixtures

(Franks and Auffret, 2007). Freezing an aqueous

pharmaceutical formulation can be conducted at a

temperature at below -35oc. The phenomena that take

place in freezing step are

5.1.1. Super-cooling

The retention of the liquid state below the equilibrium

freezing point of the solution is termed as “super-

cooling”. It always occurs during freezing often in the

range of 10-15°C or more (Searles, 2004).

Super-cooling is of two types,

5.1.1.1. Global super-cooling

It is the process in which the entire liquid volume exhibits

a similar level of super cooling

5.1.1.2. Local super-cooling

In this method, only a small volume of the liquid is super

cooled. Super-cooling is a non-equilibrium, meta-stable

state, which is similar to an activation energy necessary

for the nucleation process

5.1.2. Ice-nucleation

Due to density fluctuations from Brownian motion in the

super-cooled liquid water, water molecules form clusters

with relatively long-living hydrogen bonds (Matsumoto et

al., 2002) with similar molecular arrangements as in ice

crystals. Because this process is energetically

unfavorable, these clusters break up rapidly which results

in the formation of ice nuclei. The probability for these

nuclei to grow in both number and size is more

pronounced at lowered temperatures. Nucleation is of two

types

5.1.2.1. Homogeneous nucleation

The limiting nucleation temperature of water is referred

to as the “homogeneous nucleation temperature” that

appears to be at about -40°C. At this temperature, pure

water sample will contain at least one spontaneously

formed active water nucleus, capable of initiating ice

crystal growth.

5.1.2.2. Heterogeneous nucleation

In heterogeneous nucleation ice-like clusters are formed

via adsorption of layers of water on “foreign impurities”.


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Such “foreign impurities” may be at the surface of the

container, particulate contaminants present in the water or

even sites on large molecules such as proteins. In all

pharmaceutical solutions and even in sterile-filtered water

for injection, the nucleation observed is “heterogeneous

nucleation”.

5.1.3 Ice crystal growth

Once the critical mass of nuclei is reached, ice

crystallization occurs rapidly in the entire system which

leads to the formation of stable ice crystals. Once stable

ice crystals are formed, their growth proceeds by the

addition of molecules to the interface. As crystallization

begins, the product temperature rises rapidly to near the

equilibrium freezing point. After the initial ice network

has formed, additional heat is removed from the solution

by further cooling and the remaining water freezes when

the previously formed ice crystals grow. The number of

ice nuclei formed, the rate of ice growth and the ice

crystals size depend on the degree of super-cooling

(Rambhatla et al., 2004). The degree of super-cooling

depends on the solution properties and process

conditions, and is defined as the difference between the

equilibrium ice formation temperature and the actual

temperature at which ice crystals first form (Pikal et al.,

2002). Ice crystal growth is controlled by the latent heat

release and the cooling rate at which the sample is

exposed to. The temperature drops when the freezing of

the sample is completed. The cooling rate can be

represented by the following equation

T - temperature is a function of time (t) and location (r).

The cooling rate during freezing determines the size and

structure of ice crystals and pores in the lyophilized

product. Fast freezing leads to a large number of small ice

crystals resulting in high product resistance to vapor flow

and therefore extensive primary drying times. This effect

can be at least partially compensated by performing

thermal treatment following the freezing step also

referred to as “Annealing”. The frozen product is heated

up to a temperature below the eutectic melting point but

above the Tg of the amorphous phase, resulting in growth

of ice crystals and formation of an extended pore

structure (Searles et al., 2001a). Additionally, quantitative

crystallization of crystallizable solutes is facilitated,

which is especially important in the case of mannitol to

avoid vial breakage. Alternatively, the nucleation

temperature can be controlled using nucleation agents

(i.e. deliberately added foreign particles) or an electric

field (Searles et al., 2001a). Another possibility is the ice

fog technique which includes purging of the chamber

with very cold nitrogen, causing moisture in the air to

crystallize and initiate nucleation of ice at a specified

product temperature.

5.2. Primary Drying

It is characterized by receding boundary layer of ice in

the vial. This step traditionally is carried out at chamber

pressures of 40-400 Torr and shelf temperatures ranging

from -30°C to -10° C. In this phase the chamber pressure

is reduced up to 0.01 to 0.1mbar by introducing vacuum

in to the product chamber. Heat is applied to the product

to cause the frozen mobile water to sublime. The water

vapor is collected on the surface of a condenser. The

condenser must have sufficient surface area and cooling

capacity to hold all the sublimed water from batch at a

temperature lower than the product temperature. If the

temperature of the ice on the condenser is warmer than

the product, water vapor will tend to move towards the

product, and the drying will stop.The sublimation rate can

be modeled by the following equation

------------2

Ap - cross sectional area of the product, Pp - vapor

pressure of the product at the sublimation front, Po -

partial vapor pressure in the product vial and Rp -

resistance of the dried product layer to vapor flow.

Throughout this stage, the product is maintained in the

solid state below the collapse temperature of the product

in order to dry the product with retention of the structure

established in the freezing step. The driving force for

sublimation of ice during primary drying is the pressure

difference between the vapor pressure of ice at


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sublimation front and the partial pressure of water vapor

in the freeze dry chamber. Since vapor pressure is related

to temperature, it is necessary that the product

temperature is warmer than the condenser temperature.

The molecules of water move from higher vapor pressure

region in the chamber towards the lower vapor pressure

region in the condenser. It is extremely important that the

temperature at which a product is freeze dried is balanced

between the temperature that maintains the frozen

integrity of the product and the temperature that

maximizes the vapor pressure of the product. This

balance is key to optimum drying. The condenser is kept

at a low temperature, generally around –60ºC. The

temperature of the product should be kept as close to the

glass transition temperature as possible for maximum

efficiency in drying.

The drying rate and the heating rate are critical during

this phase. If the drying proceeds too rapidly, the dried

product can be displaced out of the container by escaping

water vapor.

It is an important that the product temperature does not go

higher than the Tg, as this can cause the product to

collapse (Fig 1.0).

Figure 1.0 Normal Product (left) Collapsed Product (right).

The collapse (Collapse is a change in the morphology,

solubility and chemical integrity when molecules change

back into the liquid state) temperature is the glass

transition temperature (Tg) in the case of amorphous

products or the eutectic temperature (Te) for crystalline

products. This will cause degradation of the product and

change the physical characteristics of the dried material,

make it harder to reconstitute and visually unappealing.

This is accomplished by being at a low temperature and

pressure and then increasing the temperature or lowering

the pressure to go directly to the vapor phase as indicated

on the phase diagram below. In freeze drying, the

temperature of the product is increased at constant

pressure (Fig 2.0).

Figure 2.0 Phase Diagram

The temperature difference between chamber and

condenser and pressure difference between solution in

vials and vacuum pump drives ice out of vial and on to

the condenser. At the end of primary drying stage, the

sublimation rate will be significantly reduced, indicating

that there is not much frozen water left in the product.

The product cools after sublimation of water, and remains

colder than the shelf temperature. When all of the ice has

sublimed, the product temperature will approach the shelf

temperature and this signals the beginning of secondary

drying (Fig 3.0).

Figure 3.0 Freezing, primary drying, secondary drying

5.3. 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%. Therefore, 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. This desorption is


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used to remove water of crystallization, randomly

dispersed water molecules in a glassy material,

intracellular water, and absorbed water. This step is

accomplished by raising the shelf temperature to higher

than ambient conditions. The shelf temperature can be

raised to 15-300C, for allowing the water molecules to

desorbs under vacuum. The shelf temperature should not

be raised above the product temperature; otherwise

degradation of the product occurs. The product may

appear to be dry at the end of the primary drying stage

but, the moisture content may still be 7-8% weight.

Secondary drying continues until the desired moisture

content of the product is achieved. The moisture content

should be less than or equal to 2% of the product weight.

The product should not be over dried, and should not

have final moisture content below 1.5 %weight in order

to preserve the cake structure. Some chemotherapeutics

and antibiotics can have moisture contents as low as 0.1

weight%. Secondary drying parameters are based on the

quantity and nature of residual water in the product and

the absorption, adsorption and desorption processes. It is

also important to know how much heat the product can

withstand without degrading and the shelf temperature

should not be raised above this temperature. In Figure 4,

secondary drying, along with freezing and primary

drying, is shown in a typical graph of product, condenser

and shelf temperatures versus time. The product

temperature closely follows slightly below the shelf

temperature as the water is being desorbed. The

condenser remains at a low temperature throughout the

entire process.

Figure 4.0 Process of lyophilization.

6. TYPICAL LYOPHILIZATION PROCESS

The first step that takes place in lyophilization process is

component preparation i.e. the sterile solution should be

prepared,compound, mixed, filtered. The filtered solution

is filled into containers (vials). Partially insert a special

designed rubber closure onto the vials. Aseptically load

the vials into a freeze dry chamber. Freeze every single

solution in every vial below a pre-determine critical

temperature. Using appropriate application of temperature

and pressure, sublime the ice from the product. Using

further application of temperature and pressure, remove

the necessary amount of bound water from the product.

Automatically stopper the vials, neutralize the chamber.

Aseptically remove the vials from the chamber and apply

aluminum seals. The process of lyophilization is shown

diagrammatically in Fig 5.0.

Figure 5: Lyophilization review process.

7. DESIGN OF LYOFREEZER (Snowman, 2006)

The Essential Components of lyofreezer include

7.1 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. The door locking

arrangement by a hydraulic or electric motor for pressure

vessels.

7.2 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


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to perform. The shelf act as a heat exchanger, removing

energy from the product during freezing, and supplying

energy to the product during the primary and secondary

drying segments of the freeze drying cycle. 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.

7.3 Process Condenser

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 condense will consist of coils or

sometimes plates which are refrigerated to a low

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. Hence there is

designation “external condenser” and “internal

condenser”. Physically, the external condenser is

traditionally placed behind the chamber, but it may be at

the side, below or above. The position of the condenser

does not affect trapping performance. For an internal

condenser the refrigerated coils or plates are placed

beneath the shelves on smaller machines, and behind the

shelves on larger machines, but again there is no

performance constraint, only the geometry of the

chamber.

7.4 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. 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.

7.5 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. The cooling

energy is supplied by compressors or sometimes-liquid

nitrogen. 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

7.6 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.

7.7 Sensors

Temperature measuring devices used are RTDs (PT100)

or thermocouples (normally type T). Vacuum sensors

include two main types- thermoelectric or Pirani gauges

and capacitance manometers.

7.8 Control System

Control may be entirely or usually fully automatic for

production machines. The control elements required are

as mentioned above, shelf temperature and pressure plus

time. A control program will set up these values as

required by the product or the process. The time may vary

from a few hours to several days. Other data such as a

product temperatures and process condenser temperatures

can also be recorded and logged.

8. PRINCIPLES OF LYOPHILIZATION

The material is first frozen and transferred to a drying

chamber. During the drying stage, the material in the

chamber is subjected to high vacuum. Heat is applied

carefully to the material, and a condenser used in the

chamber is 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. The main principle that takes

place in lyophilization is as follows.

8.1. Heat transfer

Heat supplies the energy necessary for sublimation of the

water. An ice crystal is composed of pure water that is

crystal lattice. The molecules have 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


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solid and sublime, the thickness of dry outer surface of

the specimen increases, 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 (Pikal et al., 1984; Brulls and Rasmuson, 2002).

The pathways for transfer of energy through these three

mechanisms are illustrated in Fig. 6.

Figure 6.0: Types of heat transfer to the product

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. This

area 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 tubing vials designed for

lyophilization (Ku et al., 2005; Kuu et al., 2009). 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:

---------------- 3

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 dz (Pikal, 2002).

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 7.0. 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.

Figure 7.0 Resistances in Heat Transfer

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 top shelf, as well as chamber

door and walls (Rambhatla and Pikal, 2003). The warmer

surface radiates electromagnetic energy which is

absorbed by the colder surface. Although this pathway

also depends on the distance between the surfaces, the

most important parameter is the temperature difference.

Radiative heat transfer can be described by the Stefan

Boltzmann equation

(T24—T1

4 ) ----------- 4

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—T1

4 )

- difference between the temperature of the two surfaces

to the fourth power.

The effective emissivity is an important parameter for

surface materials used in the construction of a freeze


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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 regarded

during transfer and scale-up of lyophilization cycles

between freeze-dryers with different radiation

characteristics.

8.2. 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 (Kuu

et al., 2006). These pores are created when ice crystals

are removed by sublimation, and their size, shape and

interconnection are influenced by the freezing process

(Rambhatla et al., 2004). Rp values depend on the

thickness of the already dried cake layer, and change

during the course of the drying process (Pikal et al.,

1983).

Figure 8.0 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

-------- 5

However, this really becomes a moving boundary

problem, as Rp increases with time as the ice moves out

of the product cake and must be solved through numerical

methods (Carpenter et al., 1991).

8.2.1 Coupling between Heat and Mass Transfer

During the steady state of primary drying, the heat

removed by sublimation of ice is in equilibrium with the

amount of heat introduced into the product. Heat and

mass transfer during freeze-drying are coupled which can

be described by:

dQ/dt=(dm/dt)····∆HS+ms····cv(dT /dt) -------------6

dQ/dt - heat flow to the product, dm/dt - mass removal by

sublimation, ∆HS - temperature-dependent heat of

sublimation of ice (cal/g), mS - sample mass (g), cv -

specific heat of the sample (cal/K*g) and dT/dt - change

of product temperature (K/s).

The first term describes the rate of heat removal by

sublimation, the second 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 ···· ∆Hs -------------7

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 (Pikal, 1985).

In the Fig 9.0, the Schematic representation of heat and

mass transfer in the Freeze Dryer is illustrated.

Temperature difference between chamber, condenser and

pressure differential between solution in vials and

vacuum pump drives ice out of vial and onto the

condenser.


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Figure 9.0 Schematic representation of Heat and Mass Transfer in the Freeze Dryer

9. Critical Process Parameters in freeze drying.

Properties of the formulation and the design of the freeze-

drying process are closely interrelated. The critical

temperature of the product governs the maximum

allowable temperature at the sublimation interface (Tp)

during primary drying. These critical temperature is

important in freeze drying. Freeze-drying above the product

critical temperature can lead to Loss of physical structure,

Incomplete drying (high moisture content), Decreased

solubility, Reduced activity and/or stability. Freeze-drying

too far below the product critical temperature can lead to Poor

efficiency, high cost and longer cycles than necessary.

The overall goal of a freeze-drying cycle optimization is

to keep the product temperature (Tp) close to the critical

temperature during primary drying to cut cycle time. It

should be noted that the sublimation rate (dm/dt)

increases dramatically when the product temperature at

the sublimation interface increases (approximately a

factor of two for a 5°C increase in product temperature)

(Meister and Gieseler, 2006). The critical temperature is

known to be the collapse temperature (Tc) or the glass

transition temperature of the maximal freeze concentrate

(Tg ') for an amorphous and the eutectic temperature

(Teut) for a crystalline formulation. Note that Tc and Tg'

are not necessarily the same, Tc was in several cases found

to be higher (1-5°C) than Tg' which might be critical for

process optimization. However, Tc (or Tg') of an

amorphous formulation is much lower compared to a

crystalline formulation, but an amorphous phase is often

required to stabilize the drug. A common standard to

determine Tg' or Teut is differential scanning calorimetry

(DSC). Finally, there is increasing interest in evaluating

the product resistance (Rp) as a CPP.

10. Desired characteristics of a lyophilized product

A lyophilized product should possess desirable

characteristics which includes intact cake, sufficient

strength, uniform color, sufficiently dry, sufficiently

porous, short reconstitution time, sterile, maintenance of

the characteristics of the original dosage form upon

reconstitution including solution properties, structure or

conformation of proteins and particle-size distribution of

suspensions and long-term stability

11. Excipients used in lyophilized formulation

The design of a lyophilized formulation is dependent on

the requirements of the active pharmaceutical ingredient

(API) and intended route of administration. A formulation

may consist of one or more excipients that perform one or

more functions. Some freeze-dried formulations contain

API only (e.g., cephalosporins, vancomycin, antibodies)

possibly because of the relatively high content of the

active ingredient (typically 10 mg/mL or more)

(Schwegman et al., 2005). In many other cases, excipients

are needed. According to the International

Pharmaceutical Excipients Council, pharmaceutical

excipients are substances other than the

pharmacologically active drug or pro-drug which are

included in the manufacturing process or are contained in

a finished pharmaceutical product dosage form.

Excipients for lyophilization usually fit one of the

following categories: bulking agents, stabilizers,

buffering agents, tonicity modifiers, surface-active agents

or collapse temperature modifiers.

11.1. Bulking agents

Bulking agents are used to provide product elegance (i.e.,

satisfactory appearance) as well as sufficient cake

mechanical strength to avoid product blow-out. When a

very dilute solution is lyophilized, the flow of water

vapor during primary drying may generate sufficient

force on the cake to break it and carry some of it out of

the vial. Bulking agents simply function as fillers to

increase the density of the product cake (Pikal, 2002).

Amorphous excipients can serve as bulking agents, but

due to relatively low collapse temperatures most of them

require long processing times, and are not favored.

Crystalline bulking agents produce an elegant cake

structure with good mechanical properties. mannitol and


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glycine are preferred since they are crystallizing

compounds. Mannitol is by far the most commonly used

bulking agent. A formulation based on mannitol is

usually elegant, reconstitutes quickly, and is generally

easy to freeze-dry without risk of product damages ,

except for the potential of vial breakage, which can be

minimized by small fill depths, slow freezing, avoiding

freezing temperatures less than about -25°C until

crystallization is complete or annealing respectively.

However, these materials often are ineffective 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.

11.2. 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 undergoes

minimal pH change during freezing such as citrate and

histidine buffers (Williams and Dean, 1991).

11.3. Stabilizers

The most important group of stabilizers used in freeze-

drying is classified in cryo- and lyoprotectants. They

protect the API (favorably a protein) from damage during

freezing (cryoprotection) and/or dehydration

(lyoprotection) induced denaturation In liquid state

(during freezing) preferential interaction is the most

important stabilization mechanism which means that a

protein prefers to interact with either water or an

excipient in an aqueous solution. In the presence of a

stabilizer, the protein prefers to interact with water and

the excipient is preferentially excluded. Other

stabilization mechanisms include modification of the size

of ice crystals, reduction (instead of elevation) of surface

tension, increase of the viscosity of the solution

(restricting diffusion of reactive molecules) and

suppression of pH changes. 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

reduce proteins by means of the mallard reaction.

11.4. Tonicifying Agents

Tonicity modifiers (e.g., NaCl or glycerol) are

occasionally formulated in products for human use to

make the reconstituted product isotonic (e.g., for

subcutaneous or intramuscular injections) (Bhatnagar et

al., 2007). 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 in the amorphous phase (Akers et al., 1995).

Tonicity modifiers also can be included in the diluents

rather than the formulation.

Figure 10: Classification of commonly used excipients used in lyophilization (Bahetia et al., 2010).

12. APPLICATIONS OF LYOPHILIZATION

TECHNOLOGY

The main application of this dynamic freeze-drying

technology is found in the Industries.

12.1. Industrial applications

12.1.1Pharmaceutical industry

a. Antibiotics macromolecules and electrolytes are

being produced by freeze-drying.

b. Used for drying of heat sensitive products for

example: antibiotics, blood products and vaccine.

c. Development of solid protein pharmaceuticals (for

long term storage).

d. Lyophilized nasal inserts.


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e. Drying of micro and nano particles and lyosphere.

12.1.2. 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 with available water. Instant coffee is

sometimes freeze-dried, despite high costs of freeze-

driers. 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.

12.1.3. Other industries

In chemical synthesis, products are often lyophilized 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.

12.2. Other applications

Organizations such as the document conservation

laboratory at the United States National Archives and

Records Administration (NARA) have done studies on

freeze-drying as a recovery method of water damaged

books and documents. While recovery is possible,

restoration quality depends on the material of the

documents. In bacteriology freeze-drying is used to

conserve special strain. In high-altitude environments, the

low temperatures and pressures can sometimes produce

natural mummies by a process of freeze-drying.

Advanced ceramics processes sometimes use freeze-

drying to create a formable powder from a sprayed slurry

mist. It creates softer particles with a more homogeneous

chemical composition than traditional hot spray drying.

Recently, some taxidermists have begun using freeze-

drying to preserve animals, such as pets. Freeze drying is

also used for floral preservation. Wedding bouquet

preservation has become very popular with brides who

want to preserve their wedding day flowers. Some other

less common applications of lyophilization are recovery

of water-damaged books and manuscripts and

preservation of archaeological specimens, tissue for

spare-parts surgery, museum specimens for display such

as plants and animals, and vegetable matter for research

programs.

Table 3: Lyophilized products available in the market in powder form

S. No Drug Name Company Name

1 Cefaxone Lupin Pharmaceuticals Pvt Ltd 2 Cefogram Orchid Pharmaceuticals Pvt Ltd 3 Fortum Glaxo Smithkline

4 Pantoprazole Zenon Health Care, Aristo

Pharmaceuticals 5 Rebolac I.V Cadila Pharmaceuticals 6 Omez Dr.Reddys Laboratories 7 Reflin Ranbaxy 8 Rabeprazole I.V Dr.Reddys Laboratories 9 Omeprazole Neon Antibiotics 10 Tigecycline Natco Pharma Ltd 11 Cilastitatin Natco Pharma Ltd 12 Ganciclovir Natco Pharma Ltd 13 Omeprazole Natco Pharma Ltd 14 Bortezomib Natco Pharma Ltd 15 Pemetrexed Natco Pharma Ltd 16 Zoledronic acid Natco Pharma Ltd 17 Docetaxel Natco Pharma Ltd

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