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We have extensive
knowledge and expertise in the utilization of lyophilization (freeze
drying) technologies for the development, manufacture, quality control and
productivity improvement of many products, especially for the medical
device and pharmaceutical industries.
Experience
has taught us that the following are some of most important factors in this
area:
- Correct and clear
establishment of the claims, features and benefits that the freeze
dried product needs to satisfy. Among them is re-hydration, solubility
and/or dissolution rates; pharmaco-kinetic efficacy rates; potency and
stability; shelf life, etc.
- Chemical composition of
the solution or mixture to be lyophilized.
- Conditioning of the
utilized materials before preparation of the final solution or
mixture. In depth, scientific understanding of the chemical, physical
properties of each material and any possible interactions thereof is
an absolute requirement to proactively eliminating major quality,
performance and cost problems.
- Physical and chemical
properties of the bottle, tray or any other container where the
lyophilized solution or mixture will be stored from raw materials to
finished, packaged product. Incomplete cleaning or surface preparation
as well as contamination, all are capable of creating major headaches
for any manufacturer.
- Full knowledge of the
current state of the employed manufacturing equipment and process,
i.e. lyophilizer; liquid dispenser; solution/mixture preparation
equipment; training and work load of engaged personnel.
LYOPHILIZATION
What is lyophilization
Lyophilization
(freeze drying) is the removal of water from frozen material. It is an
excellent method for preserving microbes and heat-sensitive materials such
as proteins, plasma, etc.
How does lyophilization work
Water
is removed from frozen samples mainly by sublimation- water is converted
from the frozen state into vapor, thus bypassing the liquid phase. The rate
of sublimation depends on vapor pressure, which is affected by the system
vacuum and sample temperature.
The frozen sample
absorbs heat, causing water in the sample to enter the vapor phase and
migrate into the instrument atmosphere where it is removed by refreezing on
the condenser.
Drying first occurs
at the surface of the sample. As drying proceeds, water is removed from
deeper layers of the sample and the drying rate slows.
Three steps in lyophilization
- Freezing. The sample is
placed in a freezing vial/flask. The purpose is to completely freeze
the sample.
- Primary drying.
Approximately 90% of the total water in the sample (essentially all of
the free water and some of the bound water) is removed by
sublimation.
- Secondary drying. Bound
water is removed by desorption, resulting in a product that has
<1-3% residual water. This step requires 1/3 - 1/2 the time needed
for primary drying.
How is a sample frozen
There
are two freezing methods and their use depends on the type of container
holding the sample. Small volumes (less than a few milliliters) may be
placed in a vial and frozen in a regular freezer. Larger volumes (up to
about 100 ml per freezing flask) are shell frozen and dried by the manifold
method.
Shell freezing
involves rotating the flask containing the sample in a freezing bath so the
sample freezes on the walls of the flask. This method maximizes the surface
area to thickness ratio thereby facilitating water removal from the sample.
Do not freeze a
large block of sample in the bottom of the freezing flask; the sample will
be too thick for efficient water removal and may melt during drying.
Rapid freezing
results in small ice crystals which reduce drying efficiency (although it
is good for preserving structure for microscopic examination). Slower
freezing produces larger ice crystals that improve the efficiency of
drying.
For freezing
purposes, there are two kinds of samples: solutions and suspensions.
Solutions usually have water as the solvent. Solutes tend to form
eutectics, a combination of solutes that freeze at a lower temperature than
water or other solutes. The entire sample must be frozen, including the
eutectics, before the sample is ready for drying; otherwise, the unfrozen
material will expand and melt when the sample is placed under a vacuum. The
freezing temperature of eutectics is known as the eutectic temperature.
Suspensions form a
glass as they become more viscous during freezing. Eutectics do not form.
At the glass transition point the suspension forms a vitreous solid. Each
suspension has a unique glass transformation temperature. Suspensions are
very difficult to freeze dry.
Three basic
formats for freeze drying:
- Manifold drying is the
most commonly used method. Drying flasks or ampules are attached to
individual ports on a central manifold. The samples are usually frozen
by the shell method, and are quickly attached to the manifold and
placed under vacuum to prevent melting. Room temperature provides heat
for the sample. This method is useful for relatively small
volumes.
- The batch method is used
when large numbers of similar-sized containers are simultaneously
freeze dried, e.g., serum vials. A tray system is used instead of a
manifold. Heating elements in the trays supply the heat. Most batch
systems have a mechanism to seal the vials before they are exposed to
air.
- The bulk method is used
for large volumes of a single sample. The sample is poured into
special trays, frozen, and then dried in a lyophilizer. Bulk dried
samples cannot be sealed while in the instrument. Exposure to air
before packaging may affect shelf life.
Factors that affect
the efficiency of lyophilization:
- sample size
- surface area of the
sample
- thickness of the
sample
- sample
characteristics
- eutectic
temperature
- solute
concentration
- instrument factors
- condenser
temperature
- vacuum
Eutectic
temperature is the most important factor determining how much sample can be
lyophilized at one time. Vapor pressure decreases as the eutectic
temperature lowers, but the rate of heat absorption by the sample remains
the same and may cause melting. Diluting the sample with water prior to
freezing can prevent melting.
Generally, the
larger the surface area of the frozen material, the faster the rate of
lyophilization, and, conversely, the thicker the frozen material, the
slower the rate of lyophilization. Sample thickness affects the ability of
a sample to absorb and transfer heat to the surface undergoing sublimation.
Because water vapor must pass through dried material, the rate of
lyophilization in thick samples is slower, especially if the dried material
collapses onto the surface of the frozen material. Shell freezing minimizes
collapsing by increasing the surface area.
Generally, the
volume of the freeze dry flask should be 2 to 3 times that of the material
being frozen.
The condenser
must be 10-15 degree C colder than the eutectic temperature of the material
being frozen.
A vacuum of at
least 133 x 10-3m Bar is required for lyophilization.
Vapor pressure
depends on eutectic temperature and solute concentration. The vapor
pressure of water decreases as the concentration of the sample increases,
thereby slowing the rate of sublimation.
Volatile
chemicals increase the vapor pressure at the sample surface and require
less heat for sublimation, increasing the tendency to melt. These samples
may need to be diluted with water prior to freezing.
Characteristics
of the finished product
Freeze dried products
have from <1-3% residual water content and are very hygroscopic.
The stability of the
freeze-dried product depends on moisture, oxygen, and temperature. A good
seal prevents exposure of the sample to moisture and oxygen. It is best to
freeze dry samples in vials and seal them under vacuum.
Storage at high
temperatures reduces shelf life. Refrigeration or freezing is best for
long-term storage.
Contamination
of the lyophilizer
The main contaminants in
a lyophilizer are microorganisms and harmful chemicals. Microorganisms will
contaminate any freeze drying instrument unless each vial or flask is
fitted with a bacteriological filter. Cross contamination between vials is
more likely in the batch method and vials should be decontaminated upon
removal from the lyophilizer. The condenser is the most contaminated
portion of the instrument and should be decontaminated periodically.
Corrosive
chemicals and organic solvents can damage lyophilizers. Organic solvents
are generally not removed by the condenser and pass into the vacuum pump
where they mix with the pump oil, thinning the oil and damaging the pump if
the oil is not changed periodically. Corrosive chemicals can damage all
portions of a lyophilizer. Wash the system to remove these chemicals after
processing samples.
Sample
protocols
The goal is an
uncontaminated and stable microbial culture with little or no variation or
mutation. Upon reconstitution, the microorganism should exhibit the same
growth characteristics it had prior to freezing. Additives are frequently
used in suspension fluids to enhance product stability.
Bacteria: Late
logarithmic phase cultures are usually best for freeze drying. Collect
cultures grown on agar by scraping into a suspension fluid. Cultures grown
in broth are harvested by centrifugation, followed by resuspension in a
suspension fluid.
Suspension
fluids:
- meso-Inositol 5% in horse
serum
- Inositol broth 2.5%
meso-Inositol 5% in water
Note:
Inositol serum (#1) is not recommended for enterobacteria.
Fungi: Add
equal volumes of a 72-hour shake culture and a solution of 7.5% glucose in
serum. Sucrose or Inositol (5%) can be substituted for glucose. Refrigerate
immediately to prevent further replication.
Add 0.5 - 1.0
ml of suspension to a 2.0 ml freezing vial. Freeze by placing in a
laboratory freezer (-20 degrees or below). Place in lyophilizer and dry
using the batch method.
The Basics Of Freeze Drying
Freeze
Drying or Lyophilization: This is a process of stabilizing initially wet
materials (aqueous solutions or suspensions) by freezing them, then sublime
the ice while simultaneously desorbing some of the bound moisture (Primary
Drying). Following the disappearance of the ice, desorption may be
prolonged (Secondary Drying). This process is usually conducted under
vacuum.
Desorption: The release of liquids and gas
trapped within a substance.
Sublimation: Vaporization or evaporation
wholly from a solid phase without melting.
Primary Drying: Stage of freeze drying
involving the sublimation of ice, although this is usually accompanied by
concurrent desorption of bound moisture.
Secondary Drying: Prolonged drying stage (when
all visible ice is sublimed) for continued desorption until desired product
consistency.
The science of Freeze Drying
When
you need to preserve a product without altering it, there is no replacement
for freeze-drying. This gentle process removes moisture from aqueous
product, without affecting its biological, chemical or structural
properties. Because a rigid ice matrix holds the solid components in place,
the freeze drying process maintains product integrity.
Compare this with
conventional drying, which typically causes shrinkage or chemical
reactions, damaging cells and rendering an end product useless for
additional chemical analysis, or for final product display.
For many years,
freeze-drying was as much guess work and intuition as science. But you can
now count on precise, repeatable results, time after time. While the
chemistry involved in freeze drying may be complex, the process itself can
be divided into three basic process steps:
Freezing
In the first step, the product is frozen solid, which
converts the water content of the material to ice. The final temperature
must be below the product's eutectic, or collapse temperature, so that it
maintains its structural soundness.
Once
the product is frozen solid, the condenser and vacuum systems are energized
for the next critical process step.
Primary Drying
In the second stop, the objective is to remove the
unbound, or easily removed ice from the product. This water is now in the
form of free ice, which is removed by converting it directly from a solid
to a vapor, in a process called sublimation. To accomplish sublimation, a
uniform source of heat energy is applied to the ice crystals, turning them
directly into water vapor.
The
product and condenser chambers are placed under vacuum to encourage the
orderly migration of water vapor to the system's ice-collecting condenser,
and to ensure that the pressure of the water vapor remains below its
"triple point", as required for sublimation to occur.
Secondary Drying
Even after all the free ice is removed by the
sublimation process, your product may still contain enough bound water to
limit its structural integrity and shelf life.
During
secondary drying, the sorbed water, or the water that was bound strongly to
the solids in the product, is converted to vapor. This can be a slow
process; the remaining bound water has a lower pressure than free liquid at
the same temperature, which makes it difficult to remove. Secondary drying
actually starts during the primary drying phase, but must be extended after
the total removal of the free ice to achieve low enough residual moisture
levels.
Freeze-drying
is complete when all the free and bound water has been removed, resulting
in a residual moisture level that guarantees the desired biological and
structural characteristics of the final product.
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