Deepak garg M pharmacy
Lyophilization/Freeze
Drying
INTRODUCTION
Lyophilization or freeze drying is a
process in which water is frozen, followed by its 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. It is a drying process applicable to
manufacture of certain pharmaceuticals and bio logical that are thermo labile
or otherwise unstable in aqueous solutions for prolonged storage periods, but
that are stable in the dry state. The term “lyophilization” describes a process
to produce a product that “loves the dry state.
PRINCIPLE
The main principle involved in freeze
drying is a phenomenon called sublimation, where water passes directly from
solid state (ice) to the vapour state without passing through the liquid state.
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. The material to be dried is
first frozen and then subjected under a high 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 vapour
between the drying front and condenser is the driving force for removal of
water during lyophilisation.
To extract water from foods, the process
of lyophilization consists of :
1. Freezing the food so that the water
in the food becomes 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.
The fundamental process steps
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 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 separation process.
TRADITIONAL LYOPHILIZATION TECHNOLOGY
Traditional lyophilization is a complex
process that requires a careful balancing of product, equipment, and processing
techniques. For nearly 30 years, lyophilization has been used to stabilize many
types of chemical components. In their liquid form, many such biochemicals and
chemical reagents are unstable, biologically and chemically active, temperature
sensitive, and chemically reactive with one another. Because of these
characteristics, the chemicals may have a very short shelf life, may need to be
refrigerated, or may degrade unless stabilized. When performed properly, the
process of lyophilization solves these problems by putting reagents into a
state of suspended activity. Lyophilization gives unstable chemical solutions a
long shelf life when they are stored at room temperature. The process gives
product excellent solubility characteristics, allowing for rapid
reconstitution. Heat- and moisture-sensitive compounds retain their viability.
Most proteins do not denature during the process, and bacterial growth and
enzyme action, which normally occur in aqueous preparations, can be eliminated.
Thus, lyophilization ensures maximum retention of biological and chemical
purity.
PROCESSING
There are four stages in the complete
drying process: pretreatment, freezing, primary drying, and secondary drying.
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. 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.
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 vapour
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. Traditionally, lyophilization cycle design has been divided
into three parts.
1. Freezing, in which the liquid sample is cooled
until pure crystalline ice forms from part of the liquid and the remainder of
the sample is freeze-concentrated into a glassy state where the viscosity is
too high to allow further crystallization.
2. Primary drying, wherein the ice formed during the
freezing is removed by sublimation under vacuum at low temperatures, leaving a
highly porous structure in the remaining amorphous solute that is typically 30%
water. This step is carried out at pressures of 10-4 to 10-5 atmospheres, and a
product temperature of –45 to –20°C; Sublimation during primary drying is the
result of coupled heat- and mass-transfer processes.
3. Secondary drying, wherein most of the
remaining water is desorbed from the glass as the temperature of the sample is
gradually increased while maintaining low pressures. Ideally, the final product
is a dry, easily reconstituted cake with a high surface area.
LYOPHILIZATION EQUIPMENT
There are essentially three categories
of freezedryers: the manifold freeze-dryer, the rotary freezedryer and the tray
style freeze-dryer. Two components are common to all types of freezedryers: a
vacuum pump to reduce the ambient gas pressure in a vessel containing the
substance to be dried and a condenser to remove the moisture by condensation on
a surface cooled to −40 to −80°C (−40 to −112°F). The manifold, rotary and tray
type freeze-dryers differ in the method by which the dried substance is
interfaced with a condenser. In manifold freeze-dryers a short usually circular
tube is used to connect multiple containers with the dried product to a
condenser. The rotary and tray freeze-dryers have a single large reservoir for the
dried substance. 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. Manifold
freeze-dryers are usually used in a laboratory setting when drying liquid substances
in small containers and when theproduct will be used in a short period of time.
A manifold dryer 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 lower moisture content. Tray style freezedryers are typically
larger than the manifold dryers and are more sophisticated. Tray style
freeze-dryers are used to dry a variety of materials. A tray freezedryer is
used to produce the driest product for long-term storage. A tray freezedryer
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-dryers can dry products in bulk or
in vials or other containers. When drying in vials, the freeze-dryer is
supplied with a stoppering mechanism that allows a stopper to be pressed into place,
sealing the vial before it is exposed to the atmosphere. This is used for
long-term storage, such as vaccines. 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. 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 lyophilisation cycle, and minimize the amount of
moisture to permeate its walls and seal. The enclosed reagents can only remain
properly lyophilized if the container in which they are processed meats these requirements.
Lyophilization Heat Transfer
Successful lyophilization is heavily
dependent on good thermal conductivity. For this, 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.
FREEZE DRYER 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. 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 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 .
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
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. 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.
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.
Control system
Control may be entirely or usually fully
automatic for production machines. The control elements required are as
mentioned above, shelf temperature, pressure and 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.
THE FREEZE-DRYING CYCLE:
Lyophilization is the most common method
for manufacturing solid pharmaceutical products and is central to the
preservation of materials which must be dried thoroughly in order to ensure
stability. To meet this requirement, a solution’s lyophilisation occurs in
three steps: (1) freezing to convert most of the water into ice, (2)primary
drying to sublime the ice, and (3) secondary drying to remove unfrozen water by
desorption. To technically realize this manufacturing process, a freeze dryer
is commonly constructed with two main parts: a “drying” chamber holding
temperature controlled shelves is connected by a valve to a “condenser”
chamber, which contains coils capable to achieve very low temperatures between
-50°C and -80°C. The freezedrying process consists of three stages.
1) Freezing
2) Primary drying
3) Secondary drying
Freezing
Freezing is a critical step, since the
microstructure established by the freezing process usually represents the
microstructure of the dried product. The product must be frozen to a low enough
temperature to be completely solidify. 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 pre-frozen. The method of prefreezing 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, useful in preserving
structures to be examined microscopically, but resulting in a product that is,
more difficult to freeze dry.Slower cooling results in large ice crystals and
less restrictive channel in the matrix during the drying process. Products
freeze in two ways, the majority of products that are subjected to
freeze-drying consists primarily of water, the solvent and materials dissolved
or suspended in the water, the solute. Most samples that are to be freeze dried
are eutectics, which are mixtures of substances that freeze at lower
temperature than the surrounding water. This is called the eutectic
temperature. Eutectic point is the point where all the three phases’ i.e.
solid, liquid and gaseous phases coexist. It is very important in freeze-drying
to pre freeze the product to below the eutectic temperature before beginning
the freeze-drying process. The second type of frozen product is a suspension that
undergoes glass formation during the freezing process. Instead of forming
eutectics, the entire suspension becomes increasingly viscous as the temperature
is lowered. Finally the products freeze at the glass transition point forming a
vitreous solid. This type of product is extremely difficult to freeze dry.
Primary drying
After prefreezing the product,
conditions must be established in which ice can be removed from the frozen
product via sublimation, resulting in a dry, structurally intact product.
This requires very carefully control of
the two parameters.
1) Temperature and 2) Pressure involved
in freeze-drying system. The rate of sublimation of ice from a frozen product depends
upon the difference in vapor pressure of the product compared to the vapor
pressure of the ice collector. Molecules migrate from the highpressure sample
to a lower pressure area. Since vapor pressure is related to temperature, it is
necessary that the product temperature is warmer than the cold trap (ice
collector) temperature. 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 is the balance is key to optimum drying.
Primary Drying
Most products are frozen well below
their eutectic or glass transition point (point A), and the temperature is
raised to just below this critical temperature (Point B) and they are subjected
to reduced pressure. At this point the freeze-drying process is started. Vacuum
pump is an essential of a freeze drying system, and is used to lower the pressure
of the environment around the product (point C). The other essential is a
collecting system, which is a cold trap used to collect the moisture that
leaves the frozen product. The collector condenses out all condensable gases, i.e.
the water molecules and the vacuum pump removes all non condensable gases. The
molecules have a natural affinity to move toward the collector because its
vapor pressure is lower than that of the product. Therefore the collector
temperature, (Point D) must be significantly lower than the product temperature.
A third component essential in freeze-drying system is energy. Energy is
essential in the form of heat. Almost ten times, much energy is required to sublime
a gram of water from the frozen to the gaseous state as is required to freeze a
gram of water, Heat must be applied to the product to encourage the removal of
water in the form of vapor from the frozen product. The heat must be very
carefully controlled, as applying more heat than the evaporative cooling in the
system can warm the product above its eutectic or collapse temperature. Heat
can be applied by several means one method is to apply heat directly through a
thermal conductor shelf such as is used in tray drying. Another method is to
use ambient heat as in manifold drying.
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. 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. 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 favour desorption of water.
EXCIPIENTS IN LYOPHILIZED FORMULATION
The design of aq 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. Excipients may be characterized
as buffers and pH adjusters, bulking agents, stabilizers, and tonicity modifiers.
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.
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 oftren 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.
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 be reduce proteins by means of the mallard
reaction.
Tonicity adusters
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 in the amorphous phase.
Tonicity modifiers also can be included diluents rather than the formulation.
FREEZE DRYING METHODS
Three methods of freeze drying are
commonly used
Manifold method
In the manifold method, flasks ampoules
or vials are individually attached to the ports of a drying chamber. The
product either frozen in a freezer, by direct submersion in a low temperature
bath, or by shell freezing, depending on the nature of the product and the
volume to be freeze dried. The pre frozen product is quickly attached to the
drying chamber or manifold to prevent warming. The vacuum must be created in
the product container quickly, and the operator relies on evaporative cooling
to maintain the low temperature of the product. This procedure can only be used
for relatively small volumes and product with high eutectic and collapse
temperatures. Manifold drying has several advantages over batch tray drying.
Since the vessels are attached to the manifold individually, each vial or flask
has a direct path to the collector. This removes some of the competition for
molecular space created in a batch system, and is most ideally realized in a
cylindrical drying chamber where the distance from the collector to each
product vessel is the same. Heat input can be affected by simply exposing the vessels
to ambient temperature or via a circulating bath. For some products, where
precise temperature control is required, manifold drying may not be suitable.
Batch method
In a batch drying, large numbers of
similar sized vessels containing like product are placed together in a tray
dryer. The product is usually pre frozen on the shelf of the tray dryer.
Precise control of the product temperature and the amount of heat applied to
the product during drying can be maintained. Generally all vials in the batch
are treated during drying process, although some variation in the system can
occur. Slight difference in heat input from the shelf can be expressed in different
areas. Vials located in the front portion of the shelf may radiantly through
the clear door. These slight variations can result in small difference in
residual moisture. Batch drying allows closure of all vials in a lot at the same
time, under the same atmospheric condition. The vials can be stoppered in a
vacuum, or after backfiling with inert gas. Stoppering of all vials at the same
time ensures a uniform environment in each vial and uniform product stability
during storage. Batch drying is used to prepare large numbers of ampoules or
vials of one product and is commonly used in the pharmaceutical industry.
Bulk method
Bulk drying is generally carried out in
a tray dryer like batch drying. However, the product is poured into a bulk pan
and dried as a single unit. Although the product is spread through out the entire
surface area of the shelf and may be the same thickness as product in vials,
the lack of empty spaces within the product mass changes the rate of heat input
.The heat input is limited primarily to that provided by contact with the
shelf.
Technology transfer
In pharmaceutical industry “technology
transfer” refers to the processes that are needed for successful progress drug
discovery to product development to full scale commercialization.
Technology transfer can be divided in to
2 types
Tech transfer
In tech transfer, the product is
developed in labscale, subjected to scale-up, exhibit batch to full scale
commercialization.
Site transfer
In site transfer the products from the
outside customer are subjected to lab-scale feasibility trials and then to
commercial scale depending on customer need. The importance of technology transfer,
·
To gather necessary information to
transfer technology from R&D to actual
·
Manufacturing by sorting out various information
obtained during R&D.
·
To elucidate necessary information to transfer
technology of existing products between various manufacturing places.
APPLICATIONS
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 stored, shipped, and later
reconstituted to its original form for injection.
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.
Technological Industry
In chemical synthesis, products are
often freezedried 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.
Other Uses
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. In bacteriology freeze-drying is used to
conserve special strains.
The advantages of Lyophilization include
·
Chemical decomposition is minimized.
·
Removal of water without excessive
heating.
·
Enhanced product stability in a dry
state.
·
Ease of processing a liquid, simplifies
aseptic handling.
·
More compatible with sterile operations
than dry powder filling.
Disadvantages of Lyophilization include
·
Increased handling and processing time.
·
Volatile compounds may be removed by vacuum.
·
Need for sterile diluents upon
reconstitution.
