Lyophilization Freeze Drying moisture

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318 711 Lyophilization
                   :compiled by Dr. Thanaset Senawong, Biochemistry, Science KKU

What is lyophilization ?

Lyophilization is a process which extracts the water from foods and other
products so that the foods or products remain stable and are easier to store
at room temperature (ambiant air temperature).

Lyophilization is carried out using a simple principle of physics called
sublimation. Sublimation is the transition of a substance from the solid to the
vapour state, without first passing through an intermediate liquid phase. To
extract water from foods, the process of lyophilization consists of:

   1.   Freezing the food 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.

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 a 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.


The fundamental process steps are:

   1. Freezing: The product is frozen. This provides a necessary condition for
      low temperature drying.
   2. Vacuum: After freezing, the product is placed under vacuum. This
      enables the frozen solvent in the product to vaporize without passing
      through the liquid phase, a process known as sublimation.
   3. Heat: Heat is applied to the frozen product to accelerate sublimation.
   4. Condensation: Low-temperature condenser plates remove the
      vaporized solvent from the vacuum chamber by converting it back to a
      solid. This completes the seperation process.

The first step in the lyophilization process is to freeze a product to solidify all
of its water molecules. Once frozen, the product is placed in a vacuum and
gradually heated without melting the product.

This process, called sublimation, transforms the ice directly into water vapor,
without first passing through the liquid state. The water vapor given off by
the product in the sublimation phase condenses as ice on a collection trap,
known as a condenser, within the lyophilizer's vacuum chamber.

To be considered stable, a lyophilized product should contain 3% or less of its
original moisture content and be properly sealed.

Lyophilization Equipment :

A lyophilizer consists of a vacuum chamber that contains product shelves
capable of cooling and heating containers and their contents. A vacuum
pump, a refrigeration unit, and associated controls are connected to the
vacuum chamber.

Chemicals are generally placed in containers such as glass vials that are
placed on the shelves within the vacuum chamber.

Cooling elements within the shelves freeze the product. Once the product is
frozen, the vacuum pump evacuates the chamber and the product is heated.
Heat is transferred by thermal conduction from the shelf, through the vial,
and ultimately into the product.

Lyophilization Container Requirements:

The container in which a substance is lyophilized must permit thermal
conductivity, be capable of being tightly sealed at the end of the lyophilization
cycle, and minimize the amount of moisture to permeate its walls and seal.

The enclosed reagents can only remain properly lyophilized if the container in
which they are processed meets these requirements.

Lyophilization Heat Transfer:

Successful lyophilization is heavily dependent on good thermal conductivity.
For this reason, containers used in the lyophilization process must be capable
of meeting a number of heat-transfer requirements.

Such containers should be made of a material that offers good thermal
conductivity; should provide good thermal contact with the lyophilizer shelf,
which is the source of heat during processing; and should have a minimum of
insulation separating the source of heat from the product requiring heating.

Poor thermal conductivity often results from the use of containers made of
materials with low coefficients of heat transfer. It can also be caused by the
shape, size, or quality of the container.

It may come from thermal barriers, such as excessive amounts of material,
which can act as insulation, preventing energy from being transferred to the
point at which the frozen ice and dried product interface.

Poor thermal conductivity often results in a product that is not successfully
lyophilized. In a serum vial, the surface of the frozen cake must sublime first
to allow the ice vapor to escape.

Thereafter, the sublimation front moves as the drying proceeds. Generally,
the sublimation front simultaneously moves downward toward the bottom of
the serum vial and inward toward the center of the frozen cake (the core).

If sublimation is not controlled—and it cannot be controlled when significant
thermal barriers exist—then portions of the chemicals may actually be
vacuum-dried rather than freeze-dried.

The processed product will then not possess the defined and reproducible
characteristics of a properly lyophilized material, such as maximized retention
of activity, optimized shelf life, rapid reconstitution, and a consistent finished

Sealing Lyophilized Products:

A properly lyophilized product must be sealed within its container prior to
removal from the ultradry atmosphere that exists at the end of the
lyophilization cycle.

A product that has been dried to less than 3% residual moisture will, when
exposed to an environment with greater than its own moisture level, absorb

as much moisture as it can. The product's quality will immediately be

All of the desirable characteristics of a lyophilized product—such as increased
shelf life, enhanced chemical performance, and rapid reconstitution—will be
compromised. This reintroduction of moisture can lead to loss of product,
product failures in the field, false results, and even product recalls.

The most common mistake that companies make is to use packages that
cannot be sealed inside the lyophilizer prior to repressurization. For example,
the manufacturing process for some diagnostic products may require
lyophilizing the product inside a large number of screw-top tubes.

There is no practical way to seal these tubes inside of a lyophilizer prior to
terminating the batch, so the company will assemble a large production crew
to apply the tops manually—often in a room incompatible with lyophilization.

The recently stabilized chemistry will be jeopardized by exposure to
unacceptably high and variable moisture levels during the manual sealing
process. Exposing lyophilized material to atmospheric moisture in this way
may result in an unstable product.


Lyophilization has many advantages compared to other drying and preserving

   1. Lyophilization maintains food/ biochemical and chemical reagent
      quality because they remains at a temperature that is below the
      freezing-point during the process of sublimation; The use of
      lyophilization is particularly important when processing lactic bacteria,
      because these products are easily affected by heat.
   2. Food/biochemicals and chemical reagents which are lyophilized can
      usually be stored without refrigeration, which results in a significant
      reduction of storage and transportation costs.
   3. Lyophilization greatly reduces weight, and this makes the products
      easier to transport. For example, many foods contain as much as 90%
      water. These foods are 10 times lighter after lyophilization.
   4. Because they are porous, most freeze-dried products can be easily
      rehydrated. Lyophilization does not significantly reduce volume,
      therefore water quickly regains its place in the molecular structure of
      the food/ biochemicals and chemical reagents.

     Lyophilization: Freeze-Drying
        A Downstream Process
Reference: Snowman, John W. Downstream Processes: Equipment and
Techniques, pages 315-351, 1988 Alan R. Liss, Inc.

Lisa Menyhart
Introduction to Biochemical Engineering
Professor Bungay

Biological materials often must be dried to stabilize them for storage or
distribution. Drying always causes some loss of activity or other damage.
Lyophilization, also called freeze-drying, is a method of drying that
significantly reduces such damage. Because lyophilization is the most complex
and expensive form of drying, its use is usually restricted to delicate, heat-
sensitive materials of high value.

Substances that are not damaged by freezing can usually be lyophilized so
that refrigerated storage is unnecessary. (Important exceptions are
mammalian cells, nearly all of which are destroyed by lyophilized.) Many
microorganisms and proteins survive lyophilization well, and it is a favored
method of drying vaccines, pharmaceuticals, blood fractions, and diagnostics.
Some specialist food products are also lyophilized. They rehydrate easily and
quickly because of the porous structure left after the ice has sublimed. (The
word lyophilized is derived from the Greek "made solvent-loving")

Occasionally materials are lyophilized to achieve a porous, friable structure
rather than for preservation. Lyophilizers are sometimes used for
concentration of delicate materials.
The form of the product and the type of container it is to be freeze-dried in
influence the type of lyophilizer needed and how it should be operated.

       Comparison with Liquid-Phase Drying

Lyophilization gives the opportunity to avoid denaturation caused by heating
the product, by maintaining it frozen throughout drying. This is the most
obvious advantage over liquid-phase drying.

Equally important is that in liquid-phase drying there is an undesirable
shrinkage and concentration of active constituents that causes damage as
well as a movement of these constituents to the surface of evaporation,

where they form a dense, impermeable skin that inhibits drying, and later,
rehydration. Such effects can be avoided by spray drying, but this requires
brief exposure to temperatures around 100 C.

Further advantages of lyophilization for parenteral products are that the wet
material can be accurately dispensed and can be sterile filtered just before
filling into final containers so that particulate and bacterial contamination is

Thus, the principle advantages of lyophilization as a drying process are:

      Minimum damage and loss of activity in delicate heat-liable materials
      Speed and completeness of rehydration
      Possibility of accurate, clean dosing into final product containers
      Porous, friable structure

The principle disadvantages of lyophilization are:

      High capital cost of equipment (about three times more than other
      High energy costs (2-3 times more than other methods)
      Long process time (typically 24 hour drying cycle)

Lyophilization should be used when the product meets one or more of the
following criteria: unstable; heat liable; minimum particulates required;
accurate dosing needed; quick; complete rehydration needed; high value.
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.

       Principles of Lyophilization Equipment

Wet samples can be frozen by placing them in a vacuum. The more energetic
molecules escape, and the temperature of the sample falls by evaporative
cooling. Eventually it freezes. About 15% of the water in the wet material is

The simplest form of lyophilizer would consist of a vacuum chamber into
which wet sample material could be placed, together with a means of
removing water vapor so as to freeze the sample by evaporative cooling and
freezing and then maintain the water-vapor pressure below the triple-point
pressure. The temperature of the sample would then continue to fall below
the freezing point and sublimation would slow down until the rate of heat

gain in the sample by conduction, convection, and radiation was equal to the
rate of heat loss as the more energetic molecules sublimed away were

This simple approach creates numerous difficulties. When a material is frozen
by evaporative cooling it froths as it boils. This frothing can be suppressed by
low-speed centrifugation. Centrifugation also helps to dry faster by reducing
material thickness and exposing a greater surface area.

An alternative is to freeze the material before it is placed under vacuum. This
is commonly done with small laboratory lyophilizers where material is frozen
inside a flask. The flask is then attached to a manifold connected to the ice
condenser. To speed the process the material can be shell-frozen by rotating
the flask in a low-temperature bath, giving a large surface area and small
thickness of material.

For larger-scale equipment it is usual to place the material on product-support
shelves inside the drying chamber, which can be cooled so that the material is
frozen at atmospheric pressure before the vacuum is created. Without a
controlled heat in[put to the sample its temperature would fall until drying
was virtually at a standstill. For this reason it is usual to arrange a heat supply
to the product-support shelves so that, after their initial use for freezing the
product, they can be used to provide heat to replace the energy lost with the
subliming water vapor and maintain the product at a constant low

One milliliter of ice produces more than 1,000,000 ml. of water vapor at
typical lyophilization cycle pressures. The more energy-efficient vacuum
pumps cannot handle large quantities of water vapor. For this reason it is
usual to fit a refrigerated trap (called the ice condenser) between the
lyophilization chamber and the vacuum pump. Modern lyophilizers incorporate

The most important are listed below:

      Separated drying chamber and ice condenser to reduce cross-
      Provision of an isolation valve between chamber and ice condenser to
       allow for end-point determination and simultaneous loading and
      Construction of the chamber and ice condenser as pressure valves to
       allow for steam sterilization at 121 C or higher
      Cooling and heating of the product -support shelves by a circulating
       intermediate heat-exchange fluid to give even and accurate
      Additional instruments to control, monitor, and record process

   Movable product-support shelves to close the slotted bungs used in
    vials and to facilitate cleaning and loading
   Automatic control system with safety interlocks and alarms, duplicated
    vacuum pumps, refrigeration systems, and other moving parts to
    enable drying to proceed without endangering the product in the event
    of mechanical breakdown

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